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1.
Cell Stress Chaperones ; 22(6): 811-822, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28608263

RESUMO

Neoadjuvant (or induction) chemotherapy can be used for cervical cancer patients with locally advanced disease; this treatment is followed by radical surgery and/or radiation therapy. Cisplatin is considered to be the most active platinum agent drug for this cancer, with a response rate of 20%. In order to understand how the cisplatin treatment affects the stress response, in this work, we performed an exploratory study to analyze a number of stress proteins before and after cisplatin neoadjuvant chemotherapy. The study involved 14 patients; the pre- and post-chemotherapy paired biopsies were examined by hematoxylin and eosin staining and by immunohistochemistry. The proteins evaluated were p53, P16/INK4A, MSH2, nuclear protein transcriptional regulator 1 (NUPR1), and HSPB1 (total: HSPB1/t and phosphorylated: HSPB1/p). These proteins were selected because there is previous evidence of their relationship with drug resistance. The formation of platinum-DNA adducts was also studied. There was a great variation in the expression levels of the mentioned proteins in the pre-chemotherapy biopsies. After chemotherapy, p53 was not significantly affected by cisplatin, as well as P16/INK4A and MSH2 while nuclear NUPR1 content tended to decrease (p = 0.056). Cytoplasmic HSPB1/t expression levels decreased significantly following cisplatin therapy while nuclear HSPB1/t and HSPB1/p tended to increase. Since the most significant changes following chemotherapy appeared in the HSPB1 expression levels, the changes were confirmed by Western blot. The platinum-DNA adducts were observed in HeLa cell in apoptosis; however, in the tumor samples, the platinum-DNA adducts were observed in morphologically healthy tumor cells; these cells displayed nuclear HSPB1/p. Further mechanistic studies should be performed to reveal how HSPB1/p is related with drug resistance. When the correlations of the markers with the response to neoadjuvant chemotherapy were examined, only high pre-chemotherapy levels of cytoplasmic HSPB1/p correlated with a poor clinical and pathological response to neoadjuvant cisplatin chemotherapy (p = 0.056) suggesting that this marker could be useful opening its study in a larger number of cases.


Assuntos
Biomarcadores Tumorais/genética , Cisplatino/efeitos adversos , Proteínas de Choque Térmico HSP27/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Adulto , Idoso , Cisplatino/administração & dosagem , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Proteínas de Choque Térmico , Humanos , Pessoa de Meia-Idade , Chaperonas Moleculares , Terapia Neoadjuvante/efeitos adversos , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
2.
Histochem Cell Biol ; 147(6): 759-769, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28191619

RESUMO

Experimental hypothyroidism retards mammary carcinogenesis promoting apoptosis of tumor cells. ß-catenin plays a critical role in cell adhesion and intracellular signaling pathways conditioning the prognosis of breast cancer. However, the mechanistic connections associated with the expression of ß-catenin in thyroid status and breast cancer are not known. Therefore, we studied the relationship between the expression and localization of ß-catenin and apoptosis in mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) in hypothyroid (Hypot) and euthyroid (EUT) rats. Female Sprague Dawley rats were treated with a dose of DMBA (15 mg/rat) at 55 days of age and were then divided into two groups: HypoT (0.01% 6-N-propyl-2-thiouracil in drinking water, n = 54) and EUT (untreated control, n = 43). Latency, incidence and progression of tumors were determined. At sacrifice, tumors were obtained for immunohistological studies and Western Blot. The latency was longer (p < 0.05), the incidence was lower (p < 0.0001) and tumor growth was slower (p < 0.01) in HypoT rats compared to EUT. The expression of Bax, cleaved caspase-9 and caspase-3 was significantly higher in tumors of HypoT than in EUT (p < 0.05) indicating the activation of the intrinsic pathway. In this group, ß-catenin was expressed in the plasma membrane and with less intensity, while its expression was nuclear and with greater intensity in the EUT (p < 0.05). Moreover, the expression of survivin was reduced in tumors of HypoT rats (p < 0.05). In conclusion, decreased expression of ß-catenin and its normal location in membrane of mammary tumors are associated with augmented apoptosis via activation of the intrinsic pathway in HypoT rats.


Assuntos
Apoptose , Progressão da Doença , Hipotireoidismo/metabolismo , Neoplasias Mamárias Animais/metabolismo , beta Catenina/metabolismo , Animais , Feminino , Hipotireoidismo/induzido quimicamente , Propiltiouracila , Ratos , Ratos Sprague-Dawley
3.
Cancer Lett ; 372(1): 10-23, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26718258

RESUMO

Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Lactonas/farmacologia , Melanoma Experimental/tratamento farmacológico , Sesquiterpenos/farmacologia , Carga Tumoral/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Feminino , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Survivina , Fatores de Tempo
4.
Science ; 349(6243): 95-8, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26044299

RESUMO

Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Vírus da Leucemia Bovina/química , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Bovinos , Cristalografia por Raios X , Vírus da Leucemia Bovina/genética , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Estrutura Secundária de Proteína
5.
Thyroid ; 24(6): 1040-50, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24684177

RESUMO

BACKGROUND: Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. METHODS: Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. RESULTS: TRα1, TRα2, and TRß1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRß1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRß1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. CONCLUSIONS: This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.


Assuntos
Corpo Lúteo/metabolismo , Período Pós-Parto/metabolismo , Receptores dos Hormônios Tireóideos/biossíntese , Animais , Feminino , Hormônio Luteinizante , Gravidez/metabolismo , Progesterona/biossíntese , Prolactina , Propiltiouracila/farmacologia , Isoformas de Proteínas/biossíntese , RNA Mensageiro/metabolismo , Ratos Wistar , Tireotropina/biossíntese , Tri-Iodotironina/farmacologia
6.
Artigo em Espanhol | LILACS | ID: lil-698683

RESUMO

Antecedentes: La periodontitis es una enfermedad inflamatoria infecciosa que involucra una respuesta inmune del hospedero y se caracteriza por destrucción del hueso alveolar, el objetivo del estudio es analizar la expresión de citoquinas Th17 y su correlación con periodontopatógenos y el área periodontal inflamada en pacientes con periodontitis crónica. Método: Se realizó un estudio descriptivo exploratorio en el que se reclutaron 23 pacientes con diagnóstico de periodontitis crónica y un grupo control de 10 individuos sano/gingivitis. A todos los sujetos se les realizó un examen periodontal completo. Además, se utilizó el método PISA (Periodontal Inflamed Surface Area) para cuantificar el tamaño de la herida periodontal. Se recolectaron muestras de FGC, plasma y placa bacteriana para su análisis mediante técnica de ELISA de IL-17 A, IL-6, IL-23 y IL-10 y PCR para la determinación de la presencia de: P. gingivalis, T. denticola, T. forsythensis, A. actinomycetemcomitans, F. nucleatum y P. intermedia. Los datos fueron analizados utilizando estadística descriptiva y la asociación entre variables se estimó a través de modelos de regresión logística. Resultados: Se observó una tendencia al aumento, no significativa, de los niveles de IL-17A, IL-6 y IL-23 a nivel de FGC en los sujetos con periodontitis crónica (p=0.716, 0.784, 0.421, respectivamente). Los pacientes con periodontitis crónica presentaron una disminución de la IL-10 (p=0.012) y los niveles de IL-17A se correlacionaron positivamente con el área periodontal inflamada (p=0.004). A nivel de los patógenos periodontales, se observó una asociación entre la presencia de: P. gingivalis, T. denticola, T. forsythensis y los niveles de IL-6 plasmática (p=0.017, 0.033, 0.024, respectivamente).


Objective: Periodontitis is an infectious and inflammatory disease that involves a host immune response and is characterized by alveolar bone destruction. Aim: Analyze the expression of Th17 cytokines and their correlation with periodontopathogens and periodontal inflamed area in patients with chronic periodontitis. Method: A case control study was performed. At the time of delivery, 23 cases of patients with periodontal diagnosis were enrolled in the study and 10 controls with gingivitis. The diagnosis involved a complete periodontal examination with periodontal Florida probe. Also we used the PISA (Periodontal Inflamed Surface Area) index to classify the groups. Plasma and GCF samples were collected and studied for protein expression by ELISA assays for IL17A, Il6, IL23 and IL10. Plaque was analyzed by PCR for the determination of the presence of: P. gingivalis, T. denticola, T. forsythensis, F. nucleatum, P. intermedia and A. actinomycetemcomitans. Data was analyzed using descriptive statistics and the association between variables was estimated through logistic regression models. Results: There is a trend of increased GCF levels of IL17A, IL6, and IL23 with no significance. However there was an association between gingivitis and IL10 plasma and GCF levels (p=0.012). In relation to the periodontal wound size, a correlation was observed between the levels of IL6 and IL10 in GCF. Analysis of periodontal pathogens, showed an association between the presence of: P. gingivalis, T. denticola, T. forsythensis and plasma levels of IL-6 (p=0.017, 0.033 and 0.024, respectively).


Assuntos
Humanos , Masculino , Adulto , Feminino , Pessoa de Meia-Idade , /fisiologia , Periodontite Crônica/imunologia , Periodontite Crônica/metabolismo , Periodontite Crônica/microbiologia , Bactérias/isolamento & purificação , Bactérias/genética , Ensaio de Imunoadsorção Enzimática , Epidemiologia Descritiva , Líquido do Sulco Gengival , Interleucinas/fisiologia , Modelos Logísticos , Índice Periodontal , Reação em Cadeia da Polimerase
7.
Cell Stress Chaperones ; 18(5): 559-67, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23397229

RESUMO

In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of ß-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of ß-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.


Assuntos
Caveolina 1/metabolismo , Neoplasias Mamárias Animais/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , beta Catenina/metabolismo , Animais , Caveolina 1/genética , Feminino , Humanos , Imuno-Histoquímica , Células MCF-7 , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/genética , beta Catenina/genética
8.
J Periodontal Res ; 48(3): 302-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23035752

RESUMO

AIM(S): To explore the relationship between biomarkers of systemic inflammation in plasma and gingival crevicular fluid in early pregnancy and the subsequent development of pre-eclampsia in patients with periodontitis. MATERIALS AND METHODS: A case-control study was performed. From a cohort composed of 126 pregnant women, 43 normotensive healthy pregnant women were randomly selected, and 11 cases of preeclampsia were identified. Plasmatic and gingival crevicular fluid (GCF) samples were collected in early pregnancy (11-14 wk gestation). The levels of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were measured in the plasma and GCF samples, whereas the level of C-reactive protein (CRP) was measured in plasma samples. Biomarkers were determined by ELISA assays. The data were analysed using descriptive statistics, and the association between variables was estimated through logistic regression models. RESULTS: There was observed an association between pre-eclampsia and plasmatic levels of CRP (OR: 1.07; p = 0.003). Additionally, pre-eclampsia also was associated with IL-6 levels in GCF samples in early pregnancy (OR: 1.05; p = 0.039). A multiple logistic regression model suggests that increased levels of IL-6 in GCF (OR = 1.06; p = 0.02; CI 95% 1.007-1.117) in early pregnancy increase the risk of developing pre-eclampsia. CONCLUSION(S): Pregnant women with periodontitis who later development pre-eclampsia, shows increased levels of IL-6 in GCF and CRP in plasma during early pregnancy. Periodontal disease could contribute to systemic inflammation in early pregnancy via a local increase of IL-6 and the systemic elevation of CRP. Therefore, both inflammatory markers could be involved in the relationship between periodontal disease and pre-eclampsia.


Assuntos
Proteína C-Reativa/metabolismo , Líquido do Sulco Gengival/química , Interleucina-6/metabolismo , Periodontite/complicações , Pré-Eclâmpsia/etiologia , Primeiro Trimestre da Gravidez/metabolismo , Adulto , Proteína C-Reativa/análise , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-6/análise , Interleucina-6/sangue , Modelos Logísticos , Periodontite/sangue , Periodontite/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , Primeiro Trimestre da Gravidez/sangue , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
9.
Cell Stress Chaperones ; 17(6): 779-90, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22806482

RESUMO

In oligodendrogliomas, 1p loss of heterozygosity (LOH) is a predictor of good prognosis and treatment response. In contrast, in uveal melanomas, LOH of chromosome 3 has been linked to poor prognosis and downregulation of Hsp27. In the present study, we have analyzed the expression of heat-shock proteins (Hsps) to characterize subtypes of gliomas and their histopathologic features and to correlate with other molecular markers including LOH of 1p. Biopsies from patients with primary gliomas (n = 65) were analyzed by immunohistochemistry, chromogenic in situ hybridization and fluorescent in situ hybridization and methylation-specific PCR (MSP). Elevated Hsp27 and total Hsp70 expression levels were associated with high-grade astrocytomas (p = 0.0001 and p = 0.01, respectively). In grade III oligodendrogliomas, the Hsp27 levels were significantly higher (p = 0.03). Low O6-methylguanine-DNA methyltransferase (MGMT) expression was associated with grade II astrocytomas. Elevated ß-catenin expression was associated with grade III/IV astrocytomas (p = 0.003); p53 (+) tumors were more frequently found in grade III/IV astrocytomas (p = 0,001). LOH on 1p was associated with oligodendroglial tumours. In addition, a higher Hsp27 expression correlated with LOH of 1p (p = 0.017); this was also tested in two glioma cell lines. MSP was successful in only six samples. No significant correlations were found for the other markers. In conclusion, in oligodendroglial tumors, Hsp27 appeared as a surrogate marker of LOH of 1p which could also help to predict the disease prognosis. In gliomas, p53, Hsp27, Hsp70, MGMT, and ß-catenin correlated with histopathological characteristics, suggesting that these markers could predict the disease outcome and the response to treatments.


Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Perda de Heterozigosidade , Oligodendroglioma/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 1 , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Oligodendroglioma/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto Jovem , beta Catenina/metabolismo
10.
Curr Mol Med ; 12(9): 1183-97, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22804241

RESUMO

The importance of HSPs themselves in antigen presentation and cross-presentation remains controversial. Most studies agree that as part of their molecular chaperone function, HSPs can bind and present tumor associated antigens to professional antigen presenting cells through MHC class I and class II molecules, leading to the activation of anti-tumor CD8+ and CD4+ T cells. The regulation of the innate and adaptive immune responses by HSPs is still a matter of intense research. HSPs are seen as important anticancer vaccine adjuvants. They are used through different delivery systems: HSPs/antibodies, peptide/protein-HSP complexes, tumor antigen/HSP gene fusion, viral peptides/HSP complexes or gene fusion, viral proteins/bacterial HSP fusion. In preclinical models different administration routes, subcutaneous, intradermal, intramuscular or even peroral (under special conditions) can be used, and the animal toxicities are non-significant. The HSP-based vaccines can induce specific and non-specific cellular immune responses all of which are important to induce tumor rejection. In addition, the antibodies generated after vaccination are emerging as important protagonist in the antitumoral response. This response is significantly enhanced when the suppressive tumor microenvironment and the immune suppressing effector cells are blocked. Several clinical studies have been carried out and are ongoing, immunizing cancer patients with autologous tumor derived HSP-peptide complexes (HSPPCs). The most promising results have been observed in patients with melanoma and renal clear cell cancer without advanced disease. There are clinical trials with HSP-based anticancer vaccines other than with HSPPCs (including patients with non-Hodgkin lymphoma, high-grade transitional cell carcinoma of the bladder, high-grade cervical dysplasia, etc).


Assuntos
Vacinas Anticâncer/uso terapêutico , Proteínas de Choque Térmico/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Adjuvantes Imunológicos/metabolismo , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Humanos , Modelos Biológicos
11.
Int J Hyperthermia ; 28(3): 191-201, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22515340

RESUMO

PURPOSE: The objective of the present study was to examine the consequences of a mild hyperthermia in human tumour cell lines deficient and proficient in the DNA mismatch repair system (MMR) to advance our understanding on the relationship between MMR and heat shock proteins (HSPs). MATERIALS AND METHODS: The human colon carcinoma cell lines HCT116 (parent cells), HCT116 + ch2 (MMR-deficient), and HCT116 + ch3 (MMR-proficient) were used. Cells were incubated at 41°C and 42°C for 1 h and then at 37°C for 4 and 24 h. The expression of Hsp27 and Hsp72 was evaluated by immunocytochemistry. Hsp27, Hsp72, hMLH1 and hMSH2 levels were assessed by western blotting in nuclear and cytoplasmic fractions. The alkaline comet assay was used to evaluate the DNA damage. RESULTS: The mild hyperthermia significantly increased the protein expression levels of Hsp27 and Hsp72 in all cell lines, which was higher in the cytoplasm and nucleus of HCT116 + ch3 cells. We also observed that heat induced translocation of hMLH1 and hMSH2 proteins from the nucleus to the cytoplasm in HCT116 + ch3 cells. The comet assay revealed that HCT116 parent cells were more resistant to heat-induced DNA damage. However, the MMR-proficient and deficient cell lines repaired the DNA damage at the same rate. CONCLUSIONS: The present study demonstrates that hyperthermia induced the nuclear accumulation of Hsp27 and Hsp72 and affected the subcellular localisation of hMLH1 and hMSH2 in HCT116 + ch3 cells. Our findings suggest that the MMR system is not a direct determining factor for the different heat shock response in HCT116 cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reparo de Erro de Pareamento de DNA , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Hipertermia Induzida , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética
12.
Histochem Cell Biol ; 137(2): 187-94, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22083493

RESUMO

In a previous study, we measured caveolin-1 protein levels, both in the normal breast and in breast cancer. The study revealed no association between caveolin-1 expression in the epithelial compartment and clinical disease outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor associated strongly with reduced metastasis and improved survival. Using an animal model, we found that the onset of mammary tumors driven by Her-2/neu expression was accelerated in mice lacking caveolin-1. We have analysed the heat shock protein (Hsp) response in the tumors of mice lacking caveolin-1. In all cases, the mammary tumors were estrogen and progesterone receptor negative, and the levels of Her-2/neu (evaluated by immunohistochemistry) were not different between the caveolin-1 +/+ (n = 8) and the caveolin-1 -/- (n = 7) tumors. However, a significant reduction in the extent of apoptosis was observed in mammary tumors from animals lacking caveolin-1. While Bcl-2, Bax, and survivin levels in the tumors were not different, the amount of HSPA (Hsp70) was almost double in the caveolin-1 -/- tumors. In contrast, HSPB1 (Hsp27/Hsp25) levels were significantly lower in the caveolin-1 -/- tumors. The mammary tumors from caveolin-1 null mice expressed more HSPC4 (gp96 or grp94), but HSPC1 (Hsp90), HSPA5 (grp78), HSPD1 (Hsp60), and CHOP were not altered. No significant changes in these proteins were found in the stroma surrounding these tumors. These results demonstrate that the disruption of the Cav-1 gene can cause alterations of specific Hsps as well as tumor development.


Assuntos
Adenocarcinoma/metabolismo , Caveolina 1/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Apoptose , Chaperona BiP do Retículo Endoplasmático , Feminino , Imuno-Histoquímica , Camundongos , Receptor ErbB-2/metabolismo
13.
Artigo em Espanhol | LILACS | ID: lil-627543

RESUMO

Objetivo: Determinar si la condición clínica periodontal materna, el índice de masa corporal (IMC) y su variación durante el embarazo, se asocian con marcadores de inflamación sistémica: interleuquina-6 (IL-6), factor de necrosis tumoral alfa (TNF-) y proteína C reactiva (PCR) a nivel plasmático. Material y Método: Estudio clínico que analizó una cohorte de 56 embarazadas entre 18 y 38 años de edad (promedio 27.9 +/- 7.04 años), derivadas de la unidad de medicina perinatal en el centro de salud docente asistencial (CESA) de la Universidad de los Andes. Todas ellas consintieron voluntariamente su participación en el estudio. En cada trimestre de embarazo, se les realizó un examen periodontal completo, consistente en la determinación de los niveles de inserción clínica, profundidad al sondaje, índice de higiene y sangrado en seis sitios por cada pieza dentaria, excluyendo terceros molares. Adicionalmente, en cada control se obtuvieron muestras de sangre periférica para la determinación de los mediadores proinflamatorios mediante técnica de ELISA. El diagnóstico periodontal y los niveles de mediadores inflamatorios fueron relacionados con la variación del IMC durante el embarazo. Resultados: Las embarazadas con periodontitis crónica presentaron una mayor variación y aumento del índice de masa corporal (p=0.039) y niveles de IL-6 (p=0.026) en comparación con las mujeres embarazadas con gingivitis. Conclusiones: En esta serie de pacientes, se demostró una asociación entre el diagnóstico clínico de periodontitis durante el embarazo con un aumento plasmático de IL-6, así como también una asociación entre un aumento del IMC con los niveles plasmáticos de PCR.


Objective: To determine if maternal periodontal clinical status, body mass index (BMI) and its variation during pregnancy are associated with increased levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-) and C-reactive protein (CRP) in plasma. Material and Methods: This clinical study included a total of 56 pregnant women between 18 and 38 years old (mean 27.9 +/- 7.04 years), referred from perinatal medicine unit at health care center Universidad de los Andes. All pregnant patients consented to voluntarily participate in the study. In each trimester of pregnancy, they underwent a complete periodontal examination, involving the determination of clinical attachment levels, probing depth, bleeding and hygiene index at six sites of each tooth, excluding third molars. Additionally, in each clinical check, peripheral blood samples were taken for determination of pro-inflammatory mediators with ELISA immunoassay. Periodontal diagnosis and inflammatory mediators were the variables analyzed in relation to changes in BMI during pregnancy. Results: Pregnant women with chronic periodontitis had an increase and more variation of BMI (p=0.039) and higher levels of IL-6 (p=0.026) in comparison with pregnant women with gingivitis. Conclusions: In this series of pregnant patients the clinical diagnosis of periodontitis, with an increase in BMI are associated with increased plasma levels of IL-6 and CRP.


Assuntos
Gravidez , Índice de Massa Corporal , Complicações na Gravidez/sangue , /sangue , Periodontite Crônica/sangue , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Fator de Necrose Tumoral alfa/sangue , Gengivite/sangue , Estado Nutricional , Proteína C-Reativa/análise
14.
Horm Cancer ; 2(4): 214-23, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21761111

RESUMO

In normal embryonic fibroblasts, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) stabilizes E-cadherin/ß-catenin binding and the lack of NHERF1 expression promotes cell transformation thus acting as a tumor suppressor gene. We here tested the hypothesis that NHERF1 could act as a tumor suppressor gene in colon cancer as a mediator of estrogens' protective actions in colon carcinogenesis. We studied the expression and localization of NHERF1 and ß-catenin by immunohistochemistry in colonic tumors induced by 1,2 dimethylhidrazine (DMH) in Sprague-Dawley rats. One group of the rats treated with the carcinogen was ovariectomized (OVX) in the middle of the tumor induction, simulating a human menopausal condition. We observed a protective role of estrogens in colon cancer, as non-ovariectomized rats (DMH) had a reduced tumor area compared with the ovariectomized group (DMH + OVX; mean ± SE) 28.98 ± 4.65 vs. 67.58 ± 8.69 (p < 0.00380). Despite the lack of plasma estrogen stimulation, we found abundant expression of NHERF1 in colon tumors from ovariectomized rats. NHERF1 was mainly localized in the cytoplasm of the adenocarcinoma cells and lost the apical localization previously reported in normal colon tissue. We also detected expression of NHERF1 by western blot in the SW48, CACO-2, and HT29 colon cancer cell lines. Non-estrogenic factors in plasma or the tumor microenvironment may regulate NHERF1 expression in transformed colon epithelial cells. Further studies are required to understand the regulation of NHERF1 expression in colon cancer tissue.


Assuntos
Neoplasias do Colo/metabolismo , Genes Supressores de Tumor , Hormônios Esteroides Gonadais/sangue , Fosfoproteínas/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , 1,2-Dimetilidrazina/toxicidade , Animais , Western Blotting , Células CACO-2 , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Feminino , Células HCT116 , Humanos , Imuno-Histoquímica , Ovariectomia , Ratos , Ratos Sprague-Dawley
15.
Histochem Cell Biol ; 134(6): 623-30, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21079987

RESUMO

In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.


Assuntos
Colo/metabolismo , Estrogênios/fisiologia , Ciclo Estral/fisiologia , Fosfoproteínas/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , Útero/metabolismo , Animais , Colo/efeitos dos fármacos , Diestro/metabolismo , Estro/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Proestro/metabolismo , Ratos , Ratos Wistar , Útero/efeitos dos fármacos
16.
Lupus ; 19(3): 317-22, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19919974

RESUMO

Mesenchymal stem cells (MSCs) exert suppressive effects in several disease models including lupus prone mice. However, autologous MSC therapy has not been tested in human systemic lupus erythematosus (SLE). We evaluate the safety and efficacy of bone marrow (BM)-derived MSCs in two SLE patients; the suppressor effect of these cells in-vitro and the change in CD4+CD25+FoxP3+ T regulatory (Treg) cells in response to treatment. Two females (JQ and SA) of 19 and 25 years of age, fulfilling the 1997 American College of Rheumatology (ACR) criteria for SLE were infused with autologous BM-derived MSCs. Disease activity indexes and immunological parameters were assessed at baseline, 1, 2, 7 and 14 weeks. Peripheral blood lymphocyte (PBL) subsets and Treg cells were quantitated by flow cytometry, and MSCs tested for in-vitro suppression of activation and proliferation of normal PBLs. No adverse effects or change in disease activity indexes were noted during 14 weeks of follow-up, although circulating Treg cells increased markedly. Patient MSCs effectively suppressed in-vitro PBL function. However, JQ developed overt renal disease 4 months after infusion. MSC infusion was without adverse effects, but did not modify initial disease activity in spite of increasing CD4+CD25+FoxP3+ cell counts. One patient subsequently had a renal flare. We speculate that the suppressive effects of MSC-induced Treg cells might be dependent on a more inflammatory milieu, becoming clinically evident in patients with higher degrees of disease activity.


Assuntos
Lúpus Eritematoso Sistêmico/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Linfócitos T Reguladores/metabolismo , Adulto , Proliferação de Células , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Lúpus Eritematoso Sistêmico/fisiopatologia , Linfócitos/metabolismo , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Índice de Gravidade de Doença , Transplante Autólogo , Adulto Jovem
17.
Cell Stress Chaperones ; 13(2): 207-20, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18320359

RESUMO

The cadherin-catenin proteins have in common with heat shock proteins (HSP) the capacity to bind/interact proteins of other classes. Moreover, there are common molecular pathways that connect the HSP response and the cadherin-catenin protein system. In the present study, we have explored whether in breast cancer the HSP might interact functionally with the cadherin-catenin cell adhesion system. Beta-catenin was immunoprecipitated from breast cancer biopsy samples, and the protein complexes isolated in this way were probed with antibodies against HSP family members. We are thus the first to demonstrate a specific interaction between beta-catenin and Hsp27. However, beta-catenin did not bind Hsp60, Hsp70, Hsp90, gp96, or the endoplasmic reticulum stress response protein CHOP. To confirm the finding of Hsp27-beta-catenin interaction, the 27-kDa immunoprecipitated band was excised from one-dimensional polyacrylamide gel electrophoresis gels and submitted to liquid chromatography-tandem mass spectrometry with electrospray ionization, confirming a role for Hsp27. In addition, beta-catenin interacted with other proteins including heat shock transcription factor 1, P-cadherin, and caveolin-1. In human breast cancer biopsy samples, beta-catenin was coexpressed in the same tumor areas and in the same tumor cells that expressed Hsp27. However, this coexpression was strong when beta-catenin was present in the cytoplasm of the tumor cells and not when beta-catenin was expressed at the cell surface only. Furthermore, murine breast cancer cells transfected with hsp25 showed a redistribution of beta-catenin from the cell membrane to the cytoplasm. When the prognostic significance of cadherin-catenin expression was examined by immunohistochemistry in breast cancer patients (n = 215, follow-up = >10 years), we found that the disease-free survival and overall survival were significantly shorter for patients expressing P-cadherin and for patients showing expression of beta-catenin in the cytoplasm only (not at the cell surface). The interactions of beta-catenin with Hsp27 and with HSF1 may explain some of the molecular pathways that influence tumor cell survival and the clinical significance in the prognosis of the breast cancer patients.


Assuntos
Neoplasias da Mama/química , Caderinas/análise , Carcinoma/química , Proteínas de Choque Térmico HSP27/análise , Proteínas de Neoplasias/análise , beta Catenina/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/mortalidade , Carcinoma/patologia , Linhagem Celular Tumoral/metabolismo , Intervalo Livre de Doença , Feminino , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Estimativa de Kaplan-Meier , Neoplasias Mamárias Experimentais/patologia , Camundongos , Pessoa de Meia-Idade , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Prognóstico , Mapeamento de Interação de Proteínas , Receptor ErbB-2/análise , beta Catenina/metabolismo
18.
Mol Oncol ; 2(1): 102-11, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19383332

RESUMO

We have analyzed the predictive/prognostic value of Bcl-2 protein in breast cancer patients treated with neoadjuvant chemotherapy. One hundred and ten patients were submitted to two different chemotherapeutic regimens: a) 5-fluorouracil, adriamycin or epirubicin, and cyclophosphamide (FAC/FEC) during 2-6 cycles before surgery and 3 or 4 additional cycles of FAC/FEC after surgery (n=40) and b) doxorubicin (D) 75 mg/m(2) or epirubicin (E) 120 mg/m(2) during 4 cycles before surgery, and 6 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) after surgery (n=70). Bcl-2 expression, evaluated by immunohistochemistry, did not change significantly after chemotherapy and was not related to clinical/pathological response. In FAC/FEC group, Bcl-2 positive expression after chemotherapy correlated with better disease free survival (DFS) and overall survival (OS) (P=0.008 and P=0.001). In D/E group, Bcl-2 also correlated with better DFS and OS (P=0.03 and P=0.054) in the post-chemotherapy biopsies. An unusual nuclear localization of Bax was observed in some biopsies, but this localization did not correlate with the tumor response or outcome of the patients. We found that a high Bcl-2 expression had no predictive value but had prognostic value in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas c-bcl-2/análise , Antraciclinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Terapia Neoadjuvante/métodos , Prognóstico , Análise de Sobrevida , Resultado do Tratamento , Proteína X Associada a bcl-2/análise
19.
Cell Stress Chaperones ; 12(1): 33-43, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17441505

RESUMO

We describe an approach to produce an autologous therapeutic antitumor vaccine using hydroxyapatite (HA) for vaccinating cancer patients. The novel approach involved (1) the purification of part of the self-tumor antigens/ adjuvants using column chromatography with HA, (2) the employ of HA as a medium to attract antigen-presenting cells (APCs) to the vaccination site, and (3) the use of HA as a vector to present in vivo the tumor antigens and adjuvants to the patient's APCs. The vaccine was prepared using and combining HA particles, with at least 3 heat shock proteins (gp96 was one of them possibly with chaperoned proteins/peptides as shown in the slot blots) and with proteins from the cell membrane system (including Hsp70, Hsp27, and membrane proteins). The timing of HA degradation was tested in rats; the HA particles administered under the skin attracted macrophages and were degraded into smaller particles, and they were totally phagocytized within 1 week. In patients (n = 20), the vaccine was then administered weekly and showed very low toxicity, causing minor and tolerable local inflammation (erythema, papule, or local pain); only 1 patient who received a larger dose presented hot flashes, and there were no systemic manifestations of toxicity or autoimmune diseases attributed to the vaccine. Our study suggests that this therapeutic vaccine has shown some efficacy producing a positive response in certain patients. Stable disease was noted in 25% of the patients (renal carcinoma, breast carcinoma, and astrocytoma), and a partial response was noted in 15% of the patients (breast carcinoma and astrocytoma). The most encouraging results were seen in patients with recurrent disease; 4 patients in these conditions (20%) are disease free following the vaccine administration. However, we do not want to overstate the clinical efficacy in this small number of patients. The therapeutic vaccine tested in our study is working by activating the T-cell response as was shown in the comparative histological and immunohistochemical study performed in the pre- and postvaccine biopsy taken from a patient with inflammatory breast carcinoma. However, we cannot ruled out that the vaccine could also be producing an antibody(ies)-mediated response. In conclusion, this therapeutic vaccine based on HA ceramic particles and self-antigens can be safely administered and is showing some encouraging clinical results in cancer patients.


Assuntos
Autoantígenos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Cerâmica , Durapatita/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Adulto , Idoso , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Autoantígenos/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/síntese química , Durapatita/administração & dosagem , Feminino , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/imunologia , Humanos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias/patologia , Fagocitose , Projetos Piloto , Ratos , Ratos Sprague-Dawley , Pele/citologia , Pele/patologia
20.
Eur J Pharmacol ; 556(1-3): 19-26, 2007 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-17134695

RESUMO

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fase G2 , Lactonas/farmacologia , Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Sesquiterpenos/farmacologia , Animais , Aorta Torácica/citologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Endogâmicos WKY
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