RESUMEN
Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.
RESUMEN
Heat shock proteins (HSPs) constitute a major part of the molecular chaperone system and play a fundamental role in cell proteostasis. The HSPA (HSP70) family groups twelve highly homologous HSPA proteins. Certain HSPAs are regarded as important cancer-related proteins, prospective therapeutic targets for cancer treatment, and also as potential cancer biomarkers. Heat Shock Protein A2 (HSPA2), a testis-enriched chaperone and one of the least characterized members of the HSPA family, has recently emerged as an important cancer-relevant protein with potential biomarker significance. Nevertheless, conflicting conclusions have been recently drawn both according to HSPA2 role in cancer cells, as well as to its prognostic value. In this work we have shown that one of the serious limitations in HSPA2 protein research is cross-reactivity of antibodies marketed as specific for HSPA2 with one or more other HSPA(s). Among non-specific antibodies were also those recently used for HSPA2 detection in functional and biomarker studies. We showed how using non-specific antibodies can generate misleading conclusions on HSPA2 expression in non-stressed cancer cells and tumors, as well as in cancer cells exposed to proteotoxic stress. Our findings addressed concerns on some published studies dealing with HSPA2 as a cancer-related protein.
Asunto(s)
Anticuerpos/análisis , Proteínas HSP70 de Choque Térmico/inmunología , Neoplasias/metabolismo , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Células MCF-7 , Pronóstico , Especificidad por SustratoRESUMEN
Heat shock proteins (HSPs), a large group of highly evolutionary conserved proteins, are considered to be main elements of the cellular proteoprotection system. HSPs are encoded by genes activated during the exposure of cells to proteotoxic factors, as well as by genes that are expressed constitutively under physiological conditions. HSPs, having properties of molecular chaperones, are involved in controlling/modulation of multiple cellular and physiological processes. In the presented review, we summarize the current knowledge on HSPs in the biology of epidermis, the outer skin layer composed of stratified squamous epithelium. This tissue has a vital barrier function preventing from dehydratation due to passive diffusion of water out of the skin, and protecting from infection and other environmental insults. We focused on HSPB1 (HSP27), HSPA1 (HSP70), HSPA2, and HSPC (HSP90), because only these HSPs have been studied in the context of physiology and pathophysiology of the epidermis. The analysis of literature data shows that HSPB1 plays a role in the regulation of final steps of keratinization; HSPA1 is involved in the cytoprotection, HSPA2 contributes to the early steps of keratinocyte differentiation, while HSPC is essential in the re-epithelialization process. Since HSPs have diverse functions in various types of somatic tissues, in spite of multiple investigations, open questions still remain about detailed roles of a particular HSP isoform in the biology of epidermal keratinocytes.
Asunto(s)
Epidermis/metabolismo , Proteínas de Choque Térmico/fisiología , Queratinocitos/metabolismo , Animales , Línea Celular , Humanos , Queratinocitos/citología , Queratinocitos/patología , Ratones , RatasRESUMEN
Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP72/metabolismo , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos , Técnicas de Silenciamiento del Gen , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Proteínas del Choque Térmico HSP72/deficiencia , Proteínas del Choque Térmico HSP72/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapiaRESUMEN
BACKGROUND: Cisplatin (CDDP), a small molecule platinum-based compound, is an effective anticancer drug used against a wide range of human neoplasms. Long-term clinical use of CDDP is however limited due to the development of drug resistance and the possible incidence of serious side effects including nephrotoxicity and ototoxicity. The mechanisms underlying resistance of cells to CDDP are complex, and among them, the cytoprotective involvement of proteins referred to as Heat Shock Proteins (HSP) seems potentially important. METHODS: We searched various electronic databases including PubMed and selected the reports concerning the contribution of HSPs to CDDP resistance of cancer cells and to minimize the CDDP-induced nephrotoxicity and ototoxicity. RESULTS: This critical review of data collected so far summarizes the results on the major HSPs: HSP27/HSPB1, HSP70/HSPA1, HSP90/HSPC and GRP78/HSPA5, because only these have been the subject of the most intense research in the matter discussed here. We also provide relevant information concerning some other HSPs, namely HSPA9/mortalin, HSPA2, HSP110 and DNAJ. A possible role of HSPs in counteracting CDDP-induced neprho- and ototoxicity is mentioned. CONCLUSIONS: This review shows that no universal relationship between the levels of expression of HSPs and sensitivity of cancer cells to CDDP can be confirmed. Multiple observations indicate however that such correlation can rather manifest as a molecular or cellular context-dependent phenomenon. Thus, HSPs can be viewed as an important component of the multifactorial, complex response of cancer cells to CDDP. However, to strengthen such a conviction, more extensive studies are needed.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Proteínas de Choque Térmico/fisiología , Línea Celular Tumoral , Bases de Datos Factuales , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , HumanosRESUMEN
HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.
Asunto(s)
Diferenciación Celular , Epidermis/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Queratinocitos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Adhesión Celular , Línea Celular , Proliferación Celular , Colágeno Tipo IV/metabolismo , Epidermis/patología , Femenino , Proteínas Filagrina , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/patología , Masculino , Persona de Mediana Edad , Fenotipo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , TransfecciónRESUMEN
HSPA2 belongs to the multigene HSPA family, whose members encode chaperone proteins. Although expression and function of HSPA2 is mainly associated with spermatogenesis, recent studies demonstrated that in humans, the gene is active in various cancers, as well as in normal tissues, albeit in a cell type-specific manner. In the epidermis, HSPA2 is expressed in keratinocytes in the basal layer. Currently, the mechanisms underlying the regulation of HSPA2 expression remain unknown. This study was aimed at determining whether HIF-1 and its binding site, the hypoxia-response element (HRE) located in the HSPA2 promoter, are involved in HSPA2 regulation. As a model system, we used an immortal human keratinocyte line (HaCaT) and cervical cancer cells (HeLa) grown under control or hypoxic conditions. Using an in vitro gene reporter assay, we demonstrated that in keratinocytes HSPA2 promoter activity is reduced under conditions that facilitate stabilization of HIF-1α, whereas HIF-1 inhibitors abrogated the suppressive effect of hypoxia on promoter activity. Chromatin immunoprecipitation revealed that HIF-1α binds to the HSPA2 promoter. In keratinocytes, hypoxia or overexpression of a stable form of HIF-1α attenuated the expression of endogenous HSPA2, whereas targeted repression of HIF-1α by RNAi increased transcription of HSPA2 under hypoxia. Conversely, in HeLa cells, HSPA2 expression increased under conditions that stimulated HIF-1α activity, whereas inhibition of HIF-1α abrogated hypoxia-induced up-regulation of HSPA2 expression. Taken together, our results demonstrate that HIF-1 can exert differential, cell context-dependent regulatory control of the HSPA2 gene. Additionally, we also showed that HSPA2 expression can be stimulated during hypoxia/reoxygenation stress.
Asunto(s)
Regulación hacia Abajo/fisiología , Proteínas HSP70 de Choque Térmico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Queratinocitos/metabolismo , Regulación hacia Arriba/fisiología , Células HeLa , Humanos , Regiones Promotoras GenéticasRESUMEN
The HSPA2 gene is a poorly characterized member of the HSPA (HSP70) family. HSPA2 was originally described as testis-specific and expressed at the highest level in pachytene spermatocytes of rodents, the expression of which is not induced by heat shock. HSPA2 is crucial for male fertility. However, recent advances have shown that HSPA2 is expressed in various tumors and in certain types of somatic tissues. In this review, we summarize the current knowledge on the HSPA2 expression pattern, including information on transcriptional, translational, posttranslational, and epigenetic mechanisms which regulate HSPA2 expression. We also present and discuss the current views concerning the functions of the HSPA2 protein in spermatogenetic, somatic, and cancer cells. The knowledge of the properties of HSPA2, although limited, shows this protein as a unique member of the HSPA family. However, understanding whether this protein could become a relevant cancer biomarker or a therapeutically applicable target requires extensive further studies.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Testículo/metabolismo , Animales , Expresión Génica , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Hipocampo/metabolismo , Humanos , Masculino , Neoplasias/metabolismo , Neoplasias/patología , Elementos Reguladores de la Transcripción/genética , EspermatogénesisRESUMEN
The need to find new EGFR inhibitors for use in combination with radiotherapy in the treatment of solid tumors has drawn our attention to compounds derived from genistein, a natural isoflavonoid. The antiproliferative potential of synthetic genistein derivatives used alone or in combination with ionizing radiation was evaluated in cancer cell lines using clonogenic assay. EGFR phosphorylation was assessed with western blotting. Genistein derivatives inhibited clonogenic growth of HCT 116 cancer cells additively or synergistically when used in combination with ionizing radiation, and decreased EGFR activation. Our preclinical evaluation of genistein-derived EGFR inhibitors suggests that these compounds are much more potent sensitizers of cells to radiation than the parent isoflavonoid, genistein and indicate that these compounds may be useful in the treatment of colon cancer with radiation therapy.
Asunto(s)
Neoplasias del Colon/terapia , Receptores ErbB/metabolismo , Genisteína/farmacología , Glicósidos/farmacología , Proteínas de Neoplasias/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Anticarcinógenos/síntesis química , Anticarcinógenos/química , Anticarcinógenos/farmacología , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Genisteína/síntesis química , Genisteína/química , Glicósidos/síntesis química , Glicósidos/química , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/síntesis química , Fármacos Sensibilizantes a Radiaciones/químicaRESUMEN
BACKGROUND/AIM: It has been shown that HSPA2 protein, a testis-enriched member of HSPA/HSP70 family, is important for cancer cell growth and metastasis. However, the status of HSPA2 expression in tumors and its clinical/prognostic significance are obscure. Herein we aimed to investigate the expression of HSPA2 in various types of tumors and to determine the possible clinical and prognostic significance of HSPA2 in non-small cell lung carcinoma (NSCLC). MATERIALS AND METHODS: Tissue microarrays and postoperative NSCLC tumors were tested for HSPA2 by immunohistochemistry. RESULTS: HSPA2 is expressed in the majority of tumor histotypes. In NSCLC patients (n=85), nuclear HSPA2 expression was associated with histology, TNM staging and prognosis. High HSPA2 expression was significantly related to shorter overall survival (OS) in stage I-II patients. In multivariate analysis, high HSPA2, together with stage IIIA and male sex, were associated with shorter OS in the whole group. CONCLUSIONS: As exemplified in NSCLC the status of HSPA2 in human tumors may have certain prognostic significance.
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adulto , Anciano , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Pulmón/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias/patología , Pronóstico , Tasa de Supervivencia , Análisis de Matrices TisularesRESUMEN
Genistein, due to its recognized chemopreventive and antitumour potential, is a molecule of interest as a lead compound in drug design. Recently, we found that the novel genistein derivative, [7-O-(2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl)-(1 â 4)-(6-O-acetyl-hex-2-ene-α-D-erythro pyranosyl)genistein, named G21, induced aberrations in mitotic spindle formation. In the presented study, we investigated the properties of G21 relevant to its genotoxic activity. The inhibition of topoisomerase IIα activity was evaluated in decatenation assay and immunoband depletion assay, the covalent DNA-topoisomerase IIα complexes and histone É£H2AX were detected immunofluorescently. Genotoxic effects of the tested compounds were assessed in micronucleation assay. The presence of centromeres in the micronuclei and the multiplication of centrosomes were evaluated in fluorescence immunolabelled specimens. The inhibition of tubulin polymerization was measured spectrophotometrically. We found that both tested drugs were able to inhibit topoisomerase II activity; however, G21, in contrast to genistein, blocked this enzyme at the concentration far exceeding cytotoxic IC(50). We also found that both compounds caused micronucleation in DU 145 prostate cancer cells, but in contrast to genistein, G21 exhibited aneugenic activity, manifested by the presence of centromeres in micronuclei formed in cells treated with the drug. Aneugenic properties of G21 resulted from the inhibition of tubulin polymerization and centrosome disruption, not observed in the presence of genistein. The study supports and extends our previous observations that the mechanisms of cytotoxicity of genistein and its new glycosidic derivative-G21 are significantly different.
Asunto(s)
Aneugénicos/farmacología , Disacáridos/química , Genisteína/análogos & derivados , Genisteína/farmacología , Glicósidos/farmacología , Inhibidores de Topoisomerasa II/farmacología , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Centrómero/efectos de los fármacos , Centrómero/patología , Centrosoma/efectos de los fármacos , Centrosoma/patología , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Genisteína/química , Glicósidos/química , Histonas/metabolismo , Humanos , Masculino , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Polimerizacion , Inhibidores de Topoisomerasa II/química , Tubulina (Proteína)/metabolismoRESUMEN
Photosensitizing properties of 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) functionalized by covalent attachment of one chain of poly(ethylene glycol) (PEG) with a molecular weight of 350, 2000, or 5000 Da (p-THPP-PEG(350), p-THPP-PEG(2000), p-THPP-PEG(5000)) were studied in vitro. Dark and photo cytotoxicity of these photosensitizers delivered in solution or embedded in liposomes were evaluated on two cell lines: a human colorectal carcinoma cell line (HCT 116) and a prostate cancer cell line (DU 145), and compared with these treated with free p-THPP. The attachment of PEG chains results in the pronounced reduction of the dark cytotoxicity of the parent porphyrin. Cell viability tests have demonstrated that the phototoxicity of pegylated porphyrins is dependent on the length of PEG chain and p-THPP-PEG(2000) exhibited the highest photodynamic efficacy for both cell lines. The encapsulation into liposomes did not improve the PDT effect. However, the liposomal formulation of p-THPP-PEG(2000) showed a greater tendency to induce apoptosis in both cell lines than the parent or pegylated porphyrin delivered in solution. The colocalization of p-THPP, p-THPP-PEG(2000) and p-THPP-PEG(2000) enclosed in liposomes with fluorescent markers for lysosomes, mitochondria, endoplasmatic reticulum (ER) and Golgi apparatus (GA) was determined in the HCT 116 line. The p-THPP exhibited ubiquitous intracellular distribution with a preference for membranes: mitochondria, ER, GA, lysosomes and plasma membrane. Fluorescence of p-THPP-PEG(2000) was observed within the cytoplasm, with a stronger signal detected in membranous organelle: mitochondria, ER, GA and lysosomes. In contrast, p-THPP-PEG(2000) delivered in liposomes gave a distinct lysosomal pattern of localization.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Nanomedicina , Nanopartículas , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/metabolismo , Porfirinas/metabolismo , Neoplasias de la Próstata/metabolismo , Apoptosis/efectos de los fármacos , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Neoplasias Colorrectales/patología , Citometría de Flujo , Células HCT116 , Humanos , Cinética , Liposomas , Masculino , Microscopía Confocal , Peso Molecular , Orgánulos/metabolismo , Fármacos Fotosensibilizantes/química , Polietilenglicoles/química , Porfirinas/química , Neoplasias de la Próstata/patologíaRESUMEN
Human HSPA2 is a member of the HSPA (HSP70) family of heat-shock proteins, encoded by the gene originally described as testis-specific. Recently, it has been reported that HSPA2 can be also expressed in human somatic tissues in a cell-type specific manner. The aim of the present study was to find out whether HSPA2 can increase the resistance of somatic cells to the toxic effect of heat shock, proteasome inhibitors, and several anticancer cytostatics. We used a Chinese hamster fibroblast V79 cell line because these cells do not express the HSPA2 and cytoprotective HSPA1 proteins under normal culture conditions and show limited ability to express HSPA1 in response to heat shock and proteasome inhibitors. We established, by retroviral gene transfer, a stable V79/HSPA2 cell line, which constitutively overexpressed HSPA2 protein. The major observation of our study was that HSPA2 increased long-term survival of cells subjected to heat shock and proteasome inhibitors. We found, that HSPA2 confers resistance to bortezomib-induced apoptosis. Thus, we showed for the first time that in somatic cells HSPA2 can be a part of a system protecting cells against cytotoxic stimuli inducing proteotoxic stress.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Fibroblastos/fisiología , Proteínas HSP70 de Choque Térmico/biosíntesis , Pirazinas/farmacología , Animales , Bortezomib , Ciclo Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular , Respuesta al Choque por Frío , Cricetinae , Inhibidores de Cisteína Proteinasa/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Humanos , Leupeptinas/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Estrés FisiológicoRESUMEN
Hypoxia can influence aggressiveness of melanoma by inducing specific gene expression profiles. In our previous microarray study, we identified more than 430 hypoxia-responsive genes in the B16-F10 murine melanoma cell line in vitro. Of the genes identified, seven genes: galectin 3 (Lgals3), melanoma cell adhesion molecule (Mcam), fibronectin 1 (Fn1), signal transducer and activator of transcription 3 (Stat3), microphthalmia-associated transcription factor (Mitf), max interacting protein 1 (Max1), and non-metastatic cells 1, protein (NM23A) expressed in (Nme1) are known to be associated with melanoma, but have not yet been reported as being regulated by hypoxia in human melanoma cells. In this study, we investigated whether the expression of these genes is modulated by hypoxia in microdissected areas of experimental B16-F10 tumors in vivo, as well as in commercially available human melanoma cell lines (WM35, WM1552C, WM793B, WM278, 1205Lu, and 451Lu) exposed to hypoxic conditions in vitro. Our analysis revealed significant agreement between the in-vitro and in-vivo results showing that all genes except Mitf were hypoxia regulated in the oxygen-deprived tumor regions (P<0.05). In contrast, three genes (NME1, MXI1 and FN1) proved to be hypoxia regulated in both human and mouse melanoma cells (P<0.05). Our results link these genes, for the first time, with hypoxic microenvironment of melanoma and imply that the widely used B16-F10 melanoma experimental tumor model could be a convenient research tool for further investigation of their role in the development and course of this malignancy.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Fibronectinas/genética , Melanoma/genética , Nucleósido Difosfato Quinasas NM23/genética , Neoplasias Cutáneas/genética , Proteínas Supresoras de Tumor/genética , Animales , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Melanoma/metabolismo , Melanoma/patología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. RESULTS: We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. CONCLUSIONS: Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells.
Asunto(s)
ADN/administración & dosificación , Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Lípidos/farmacología , Liposomas/metabolismo , Fosfatidiletanolaminas/metabolismo , Animales , Línea Celular Tumoral , Ratones , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues.
Asunto(s)
Proteínas HSP70 de Choque Térmico/análisis , Análisis de Matrices Tisulares , Western Blotting , Diferenciación Celular , Glándulas Exocrinas/química , Glándulas Exocrinas/metabolismo , Femenino , Proteínas HSP70 de Choque Térmico/biosíntesis , Humanos , Inmunohistoquímica , Tejido Linfoide/química , Tejido Linfoide/metabolismo , Masculino , Sistema Urogenital/química , Sistema Urogenital/metabolismoRESUMEN
This paper describes the synthesis and antiproliferative activity of conjugates of genistein (1) and unsaturated pyranosides. Constructs linking genistein with a sugar moiety through an alkyl chain were obtained in a two-step synthesis: in a first step genistein was converted into an intermediate bearing an ω-hydroxyalkyl substituent, containing two, three or five carbon atoms, at position 7, while the second step involved Ferrier glycosylation reaction, employing glycals. Antiproliferative activity of several genistein derivatives was tested in cancer cell lines in vitro. The most potent derivative, Ram-3 inhibited the cell cycle, interacted with mitotic spindles and caused apoptotic cell death. Neither genistein nor the sugar alone were able to influence the mitotic spindle organization. Our results indicate, that conjugation of genistein with certain sugars may render the interaction of derivatives with new molecular targets.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Genisteína/farmacología , Mitosis/efectos de los fármacos , Línea Celular Tumoral , Electroforesis en Gel de Agar , Genisteína/síntesis química , Humanos , Etiquetado Corte-Fin in Situ , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Genistein, the principal isoflavone constituent of soybean, attracts much attention as a natural molecule with significant affinity towards targets of potential medicinal interest, but also as a food supplement or prospective chemopreventive agent. Since its physicochemical properties are considered suboptimal for drug development, much effort has been invested in designing its analogs and conjugates in hope to obtain compounds with improved efficacy and selectivity. The aim of this article is to summarize current knowledge about the properties of synthetic genistein derivatives and to discuss possible clinical application of selected novel compounds. Some basic information concerning chemical reactivity of genistein, relevant to the synthesis of its derivatives, is also presented.
Asunto(s)
Genisteína/análogos & derivados , Genisteína/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Genisteína/química , Genisteína/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
Synthesis of novel conjugates of the non-steroidal anti-inflammatory drug - ibuprofen with nontoxic oligo(3-hydroxybutyrate) (OHB) is described. Presented results indicate that anionic ring-opening polymerization of (R,S)-beta-butyrolactone initiated with an alkali metal salt of (S)-(+)-2-(4-isobutylphenyl)propionic acid (ibuprofen) may constitute a convenient method of conjugation of selected drugs with biodegradable OHB. Furthermore using the MTT cell proliferation assay we demonstrated that ibuprofen conjugated with OHB exhibited significantly increased, as compared to free ibuprofen, potential to inhibit proliferation of HT-29 and HCT 116 colon cancer cells. However, the conjugates of ibuprofen and OHB are less toxic as was shown in oral acute toxicity test in rats. Although the mechanism of antiproliferative activity of ibuprofen-OHB conjugates (Ibu-OHB) has to be established, we suggest that partially it can be related to more effective cellular uptake of the conjugate than the free drug. This assumption is based on the observation of much more efficient accumulation of a marker compound - OHB conjugated with fluorescein, in contrast to fluorescein sodium salt, which entered cells inefficiently. Further characterization of biological properties of the ibuprofen-OHB conjugates would provide insight into the mechanism of their antiproliferative effect and assess the potential relevance of their anticancer activity.
Asunto(s)
Ácido 3-Hidroxibutírico/farmacología , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Ibuprofeno/farmacología , Ácido 3-Hidroxibutírico/síntesis química , Ácido 3-Hidroxibutírico/química , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Ibuprofeno/síntesis química , Ibuprofeno/química , Estructura Molecular , Ratas , Ratas Wistar , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a lead compound in drug design. While multiple molecular targets for genistein have been identified, so far neither for this isoflavonoid nor for its natural or synthetic derivatives disruption of microtubules and mitotic spindles has been reported. Here we describe such properties of the synthetic glycosidic derivative of genistein significantly more cytotoxic than genistein, 7-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-(6-O-acetyl-hex-2-ene-alpha-D-erythro-pyranosyl)genistein, shortly named G21. We found that G21 causes significant mitotic delay, frequent appearance of multipolar spindles, and alteration of the interphase microtubule array.