RESUMEN
BACKGROUND: Heat Shock Proteins (HSPs), a family of genes with key roles in proteostasis, have been extensively associated with cancer behaviour. However, the HSP family is quite large and many of its members have not been investigated in breast cancer (BRCA), particularly in relation with the current molecular BRCA classification. In this work, we performed a comprehensive transcriptomic study of the HSP gene family in BRCA patients from both The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohorts discriminating the BRCA intrinsic molecular subtypes. METHODS: We examined gene expression levels of 1097 BRCA tissue samples retrieved from TCGA and 1981 samples of METABRIC, focusing mainly on the HSP family (95 genes). Data were stratified according to the PAM50 gene expression (Luminal A, Luminal B, HER2, Basal, and Normal-like). Transcriptomic analyses include several statistical approaches: differential gene expression, hierarchical clustering and survival analysis. RESULTS: Of the 20,531 analysed genes we found that in BRCA almost 30% presented deregulated expression (19% upregulated and 10% downregulated), while of the HSP family 25% appeared deregulated (14% upregulated and 11% downregulated) (|fold change| > 2 comparing BRCA with normal breast tissues). The study revealed the existence of shared HSP genes deregulated in all subtypes of BRCA while other HSPs were deregulated in specific subtypes. Many members of the Chaperonin subfamily were found upregulated while three members (BBS10, BBS12 and CCTB6) were found downregulated. HSPC subfamily had moderate increments of transcripts levels. Various genes of the HSP70 subfamily were upregulated; meanwhile, HSPA12A and HSPA12B appeared strongly downregulated. The strongest downregulation was observed in several HSPB members except for HSPB1. DNAJ members showed heterogeneous expression pattern. We found that 23 HSP genes correlated with overall survival and three HSP-based transcriptional profiles with impact on disease outcome were recognized. CONCLUSIONS: We identified shared and specific HSP genes deregulated in BRCA subtypes. This study allowed the recognition of HSP genes not previously associated with BRCA and/or any cancer type, and the identification of three clinically relevant clusters based on HSPs expression patterns with influence on overall survival.
Asunto(s)
Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/genética , Neoplasias de la Mama/mortalidad , Estudios de Cohortes , Femenino , Humanos , Modelos de Riesgos ProporcionalesRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/genética , Cisplatino/efectos adversos , Proteínas de Choque Térmico HSP27/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Adulto , Anciano , Cisplatino/administración & dosificación , Aductos de ADN/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Proteínas de Choque Térmico , Humanos , Persona de Mediana Edad , Chaperonas Moleculares , Terapia Neoadyuvante/efectos adversos , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
There is tremendous disparity in scientific productivity among nations, particularly in Latin America. At first sight, this could be linked to the relative economic health of the different countries of the region, but even large and relatively rich Latin American countries do not produce a good level of science. Although Latin America has increased the number of its scientists and research institutions in recent years, the gap between developed countries and Latin American countries is startling. The prime importance of science and technology to the development of a nation remains unacknowledged. The major factors contributing to low scientific productivity are the limited access to grant opportunities, inadequate budgets, substandard levels of laboratory infrastructure and equipment, the high cost and limited supply of reagents, and inadequate salaries and personal insecurity of scientists. The political and economic instability in several Latin America countries results in a lack of long-term goals that are essential to the development of science. In Latin America, science is not an engine of the economy. Most equipment and supplies are imported, and national industries are not given the incentives to produce these goods at home. It is a pity that Latin American society has become accustomed to expect new science and technological developments to come from developed countries rather than from their own scientists. In this article, we present a critical view of the Latin American investigator's daily life, particularly in the area of biomedicine. Too many bright young minds continue to leave Latin America for developed countries, where they are very successful. However, we still have many enthusiastic young graduates who want to make a career in science and contribute to society. Governments need to improve the status of science for the sake of these young graduates who represent the intellectual and economic future of their countries.
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Investigación/tendencias , Ciencia/tendencias , Humanos , América Latina , Investigación/economía , Ciencia/economíaRESUMEN
Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 µM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.
Asunto(s)
Cadmio/toxicidad , Proteínas de Choque Térmico HSP27/metabolismo , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Femenino , Colorantes Fluorescentes/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/farmacología , Células HeLa , Humanos , Microscopía Fluorescente , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/análisis , Ácido Peroxinitroso/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Regulación hacia Arriba/efectos de los fármacos , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
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.
Asunto(s)
Caveolina 1/metabolismo , Neoplasias Mamarias Animales/metabolismo , Virus del Tumor Mamario del Ratón/genética , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , beta Catenina/metabolismo , Animales , Caveolina 1/genética , Femenino , Humanos , Inmunohistoquímica , Células MCF-7 , Neoplasias Mamarias Animales/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/genética , beta Catenina/genéticaRESUMEN
Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.
Asunto(s)
Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Daño del ADN , Lactonas/farmacología , Sesquiterpenos/farmacología , Proliferación Celular/efectos de los fármacos , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Marcadores Genéticos , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hsp27 (HSPB1) is usually overexpressed in breast cancers affecting the disease outcome and the sensitivity of tumors to chemotherapy and radiotherapy. Hsp27 interacts with other proteins such as ß-catenin, histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is deleted in many human tumors. The PI3K/Akt signaling pathway is negatively regulated by PTEN. Hsp27 is described as a key component of the Akt signaling cascade: Akt, BAD, Forkhead transcription factors, Hsp27, mitogen-activated protein kinase kinase-3 and -6. Here, we have examined whether the downregulation of Hsp27 by siHsp27 affects the PTEN levels in the MCF-7 human breast cancer cell line. PTEN was detected with two different antibodies using western blots and immunocytochemistry. p-Akt was also evaluated by western blot. In addition, Hsp27 and PTEN were immunoprecipitated to know whether these proteins interact. Intracellular colocalization studies were carried out by confocal microscopy. A significant reduction in the Hsp27 levels was noted in the siHsp27 transfected cells. These Hsp27 downregulated cells showed a significant increased expression of PTEN. The MW 76 and 55 kDa PTEN forms were upregulated as revealed by two different antibodies. The phosphatase activity of PTEN seems to be active because p-Akt levels were reduced. Hsp27 immunoprecipitation was bringing PTEN and vice versa, these two proteins seem to interact at cytoplasmic level by FRET. Downregulation of Hsp27 stabilized PTEN protein levels. Chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) levels were not significantly influenced by Hsp27 downregulation. In conclusion, we report a novel function of Hsp27 modulating the PTEN levels in human breast cancer cells suggesting an interaction between these two molecules.
Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Regulación hacia Abajo , Femenino , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Proteínas de Choque Térmico HSP27/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Células MCF-7 , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Regulación hacia Arriba , beta Catenina/metabolismoRESUMEN
Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, this heat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participate in many of the traits that permit the malignant phenotype. Thus, cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1/HSP in malignant transformation and (3) discovering approaches to therapy based on disrupting the influence of the HSF1-controlled transcriptome in cancer.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/fisiología , Neoplasias/genética , Neoplasias/patología , Factores de Transcripción/genética , Chaperonina 60/genética , Chaperonina 60/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/radioterapia , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
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.
Asunto(s)
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Pérdida de Heterocigocidad , Oligodendroglioma/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Astrocitoma/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Cromosomas Humanos Par 1 , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Femenino , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Oligodendroglioma/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adulto Joven , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Reparación de la Incompatibilidad de ADN , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Hipertermia Inducida , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genéticaRESUMEN
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.
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Adenocarcinoma/metabolismo , Caveolina 1/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Animales , Apoptosis , Chaperón BiP del Retículo Endoplásmico , Femenino , Inmunohistoquímica , Ratones , Receptor ErbB-2/metabolismoRESUMEN
Heat shock proteins (Hsp) are molecular chaperones with the capability to interact with a wide range of other proteins and are thus often found coupled with other heat shock and non-heat shock proteins. This can be an advantage to study specific interactions between a chaperone and other proteins and to generate an antitumoral immune response. In this chapter, we present two protocols to isolate Hsp. One involves column chromatography with hydroxyapatite and the other employs immunoprecipitation with antibodies coupled to magnetic beads. In both cases, we specifically want to isolate Hsp coupled with other proteins and use the Hsp complexes as intermediaries to present the coupled peptides/proteins to the immune system, or to explore the associations of a particular Hsp with other proteins.
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Cromatografía de Afinidad/métodos , Proteínas de Choque Térmico/aislamiento & purificación , Inmunoprecipitación/métodos , Materiales Biocompatibles , Línea Celular Tumoral , Durapatita , Proteínas de Choque Térmico/química , Humanos , Dominios y Motivos de Interacción de ProteínasRESUMEN
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.
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Colon/metabolismo , Estrógenos/fisiología , Ciclo Estral/fisiología , Fosfoproteínas/biosíntesis , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Útero/metabolismo , Animales , Colon/efectos de los fármacos , Diestro/metabolismo , Estro/metabolismo , Femenino , Mucosa Intestinal/metabolismo , Proestro/metabolismo , Ratas , Ratas Wistar , Útero/efectos de los fármacosRESUMEN
The heat shock proteins (HSP) constitute a superfamily of chaperone proteins present in all cells and in all cell compartments, operating in a complex interplay with synergistic/overlapping multiplicity of functions, even though the common effect is cell protection. Several reasons explain the need for investigating HSP in prostate cancer: (1) these molecules function as chaperones of tumorigenesis accompanying the emergence of prostate cancer cells, (2) they appear as useful molecular markers associated with disease aggressiveness and with resistance to anticancer therapies including hormone therapy, radiotherapy, chemotherapy and hyperthermia, and (3) they can be used as targets for therapies. The latter can be accomplished by: (i) interrupting the interaction of HSP (mainly HSPC1) with various client proteins that are protected from degradation when chaperoned by the HSP; (ii) using the chaperone and adjuvant capabilities of certain HSP to present antigenic peptides to the immune system, so this system can recognise the prostate tumour cells as foreign to mount an effective antitumoral response; and (iii) using treatment planning models taking into account the HSP expression levels to obtain more effective therapies. In summary, the study of the HSP during tumorigenesis as well as during cancer progression, and the inclusion of treatment designs targeting HSP combined with other treatment modalities, should improve prostate cancer survival in the near future.
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Proteínas de Choque Térmico/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapiaRESUMEN
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.
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Neoplasias de la Mama/química , Cadherinas/análisis , Carcinoma/química , Proteínas de Choque Térmico HSP27/análisis , Proteínas de Neoplasias/análisis , beta Catenina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biopsia , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma/mortalidad , Carcinoma/patología , Línea Celular Tumoral/metabolismo , Supervivencia sin Enfermedad , Femenino , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Estimación de Kaplan-Meier , Neoplasias Mamarias Experimentales/patología , Ratones , Persona de Mediana Edad , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Pronóstico , Mapeo de Interacción de Proteínas , Receptor ErbB-2/análisis , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/mortalidad , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Antraciclinas/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Terapia Neoadyuvante/métodos , Pronóstico , Análisis de Supervivencia , Resultado del Tratamiento , Proteína X Asociada a bcl-2/análisisRESUMEN
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.
Asunto(s)
Autoantígenos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Cerámica , Durapatita/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Adulto , Anciano , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Autoantígenos/administración & dosificación , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/síntesis química , Durapatita/administración & dosificación , Femenino , Proteínas de Choque Térmico/administración & dosificación , Proteínas de Choque Térmico/inmunología , Humanos , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Neoplasias/patología , Fagocitosis , Proyectos Piloto , Ratas , Ratas Sprague-Dawley , Piel/citología , Piel/patologíaRESUMEN
Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 degrees C), Do or Pt (doxorubicin or cisplatin alone), and HS+Do or HS+Pt (heat shock+doxorubicin or heat shock+cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS+Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS+Do. The Hsp70 induction by Pt and HS+Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS+Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival and overall survival, and hMSH2 correlated with overall survival. The results point to the utility of these molecules and of the comet assay as possible predictive markers.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Resistencia a Antineoplásicos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Adolescente , Adulto , Antineoplásicos/uso terapéutico , Biomarcadores/análisis , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Niño , Preescolar , Cisplatino/uso terapéutico , Citoplasma/metabolismo , ADN/análisis , Daño del ADN , Reparación de la Incompatibilidad de ADN , Doxorrubicina/uso terapéutico , Femenino , Proteínas de Choque Térmico HSP27 , Proteínas HSP70 de Choque Térmico/análisis , Proteínas de Choque Térmico/análisis , Humanos , Lactante , Linfocitos/química , Linfocitos/metabolismo , Masculino , Chaperonas Moleculares , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/análisis , Proteínas de Neoplasias/análisis , Neoplasias/mortalidad , Proteínas Nucleares/análisis , PronósticoRESUMEN
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.