RESUMEN
Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status.
Asunto(s)
Antraciclinas , Tiosemicarbazonas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antraciclinas/farmacología , Antibióticos Antineoplásicos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Niño , Daño del ADN , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Humanos , Tiosemicarbazonas/farmacología , Inhibidores de Topoisomerasa IIRESUMEN
Tyrosine kinase inhibitors (TKIs) are being increasingly used to treat various malignancies. Although they were designed to target aberrant tyrosine kinases, they are also intimately linked with the mechanisms of multidrug resistance (MDR) in cancer cells. MDR-related solute carrier (SLC) and ATB-binding cassette (ABC) transporters are responsible for TKI uptake and efflux, respectively. However, the role of TKIs appears to be dual because they can act as substrates and/or inhibitors of these transporters. In addition, several TKIs have been identified to be sequestered into lysosomes either due to their physiochemical properties or via ABC transporters expressed on the lysosomal membrane. Since the development of MDR represents a great concern in anticancer treatment, it is important to elucidate the interactions of TKIs with MDR-related transporters as well as to improve the properties that would prevent TKIs from diffusing into lysosomes. These findings not only help to avoid MDR, but also help to define the possible impact of combining TKIs with other anticancer drugs, leading to more efficient therapy and fewer adverse effects in patients.
Asunto(s)
Reposicionamiento de Medicamentos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Ensayos Clínicos como Asunto , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Transportadoras de Solutos/genética , Proteínas Transportadoras de Solutos/metabolismo , Resultado del TratamientoRESUMEN
For decades, retinoids and their synthetic derivatives have been well established anticancer treatments due to their ability to regulate cell growth and induce cell differentiation and apoptosis. Many studies have reported the promising role of retinoids in attaining better outcomes for adult or pediatric patients suffering from several types of cancer, especially acute myeloid leukemia and neuroblastoma. However, even this promising differentiation therapy has some limitations: retinoid toxicity and intrinsic or acquired resistance have been observed in many patients. Therefore, the identification of molecular markers that predict the therapeutic response to retinoid treatment is undoubtedly important for retinoid use in clinical practice. The purpose of this review is to summarize the current knowledge on candidate markers, including both genetic alterations and protein markers, for retinoid resistance and sensitivity in human malignancies.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Retinoides/farmacología , Retinoides/uso terapéutico , Animales , Antineoplásicos Fitogénicos/química , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Manejo de la Enfermedad , Resistencia a Antineoplásicos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Pronóstico , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Retinoides/química , Resultado del TratamientoRESUMEN
Retinoids represent a popular group of differentiation inducers that are successfully used in oncology for treatment of acute promyelocytic leukemia in adults and of neuroblastoma in children. The therapeutic potential of retinoids is based on their key role in the regulation of cell differentiation, growth, and apoptosis, which provides a basis for their use both in cancer therapy and chemoprevention. Nevertheless, patients treated with retinoids often exhibit or develop resistance to this therapy. Although resistance to retinoids is commonly categorized as either acquired or intrinsic, resistance as a single phenotypic feature is usually based on the same mechanisms that are closely related or combined in both of these types. In this review, we summarize the most common changes in retinoid metabolism and action that may affect the sensitivity of a tumor cell to treatment with retinoids. The availability of retinoids can be regulated by alterations in retinol metabolism or in retinoid intracellular transport, by degradation of retinoids or by their efflux from the cell. Retinoid effects on gene expression can be regulated via retinoid receptors or via other molecules in the transcriptional complex. Finally, the role of small-molecular-weight inhibitors of altered cell signaling pathways in overcoming the resistance to retinoids is also suggested.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Retinoides/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Receptores de Ácido Retinoico/metabolismo , Retinoides/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The induced differentiation of tumor cells into mature phenotypes is a promising strategy in cancer therapy. In this study, the effects of combined treatment with all-trans retinoic acid (ATRA) and lipoxygenase/cyclooxygenase inhibitors were examined in two osteosarcoma cell lines, Saos-2 and OSA-01. Caffeic acid and celecoxib were used as inhibitors of 5-lipoxygenase and of cyclooxygenase-2, respectively. Changes in the cell proliferation, matrix mineralization, and occurrence of differentiation markers were evaluated in treated cell populations at intervals. The results confirmed the capability of caffeic acid to enhance the antiproliferative effect of ATRA in both cell lines. In contrast, celecoxib showed the same effect in Saos-2 cells only. Furthermore, the extension of matrix mineralization was observed after combined treatment with ATRA and celecoxib or caffeic acid. The increased expression of osteogenic differentiation markers was observed in both cell lines after the combined application of ATRA and inhibitors. The obtained results clearly demonstrate the capability of lipoxygenase/cyclooxygenase inhibitors to enhance the antiproliferative and differentiating effect of ATRA in osteosarcoma cells, although some of these effects are specific and depend on the biological features of the respective tumor or cell line.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Osteosarcoma/tratamiento farmacológico , Tretinoina/farmacología , Neoplasias Óseas/patología , Ácidos Cafeicos/farmacología , Celecoxib , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Osteosarcoma/patología , Pirazoles/farmacología , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: A detailed analysis of the expression of 440 cancer-related genes was performed after the combined treatment of medulloblastoma cells with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). The combinations of retinoids and celecoxib as a COX-2 inhibitor were reported to be effective in some regimens of metronomic therapy of relapsed solid tumors with poor prognosis. Our previous findings on neuroblastoma cells using expression profiling showed that LOX/COX inhibitors have the capability of enhancing the differentiating action of ATRA. Presented study focused on the continuation of our previous work to confirm the possibility of enhancing ATRA-induced cell differentiation in these cell lines via the application of LOX/COX inhibitors. This study provides more detailed information concerning the mechanisms of the enhancement of the ATRA-induced differentiation of medulloblastoma cells. METHODS: The Daoy and D283 Med medulloblastoma cell lines were chosen for this study. Caffeic acid (an inhibitor of 5-LOX) and celecoxib (an inhibitor on COX-2) were used in combined treatment with ATRA. The expression profiling was performed using Human Cancer Oligo GEArray membranes, and the most promising results were verified using RT-PCR. RESULTS: The expression profiling of the selected cancer-related genes clearly confirmed that the differentiating effects of ATRA should be enhanced via its combined administration with caffeic acid or celecoxib. This effect was detected in both cell lines. An increased expression of the genes that encoded the proteins participating in induced differentiation and cytoskeleton remodeling was detected in both cell lines in a concentration-dependent manner. This effect was also observed for the CDKN1A gene encoding the p21 protein, which is an important regulator of the cell cycle, and for the genes encoding proteins that are associated with proteasome activity. Furthermore, our results showed that D283 Med cells are significantly more sensitive to treatment with ATRA alone than Daoy cells. CONCLUSIONS: The obtained results on medulloblastoma cell lines are in accordance with our previous findings on neuroblastoma cells and confirm our hypothesis concerning the common mechanism of the enhancement of ATRA-induced cell differentiation in various types of pediatric solid tumors.
RESUMEN
Tyrosine kinase inhibitors (TKIs) are frequently used in combined therapy to enhance treatment efficacy and overcome drug resistance. The present study analyzed the effects of three inhibitors, sunitinib, gefitinib, and lapatinib, combined with iron-chelating agents, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) or di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). Simultaneous administration of the drugs consistently resulted in synergistic and/or additive activities against the cell lines derived from the most frequent types of pediatric solid tumors. The results of a detailed analysis of cell signaling in the neuroblastoma cell lines revealed that TKIs inhibited the phosphorylation of the corresponding receptor tyrosine kinases, and thiosemicarbazones downregulated the expression of epidermal growth factor receptor, platelet-derived growth factor receptor, and insulin-like growth factor-1 receptor, leading to a strong induction of apoptosis. Marked upregulation of the metastasis suppressor N-myc downstream regulated gene-1 (NDRG1), which is known to be activated and upregulated by thiosemicarbazones in adult cancers, was also detected in thiosemicarbazone-treated neuroblastoma cells. Importantly, these effects were more pronounced in the cells treated with drug combinations, especially with the combinations of lapatinib with thiosemicarbazones. Therefore, these results provide a rationale for novel strategies combining iron-chelating agents with TKIs in therapy of pediatric solid tumors.
RESUMEN
Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.
Asunto(s)
Células Cromafines , Neuroblastoma , Glándulas Suprarrenales/metabolismo , Animales , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Femenino , Ratones , Neuroblastoma/metabolismo , Embarazo , Serotonina/metabolismoRESUMEN
Impairment of the p53 pathway is a critical event in cancer. Therefore, reestablishing p53 activity has become one of the most appealing anticancer therapeutic strategies. Here, we disclose the p53-activating anticancer drug (3S)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (MANIO). MANIO demonstrates a notable selectivity to the p53 pathway, activating wild-type (WT)p53 and restoring WT-like function to mutant (mut)p53 in human cancer cells. MANIO directly binds to the WT/mutp53 DNA-binding domain, enhancing the protein thermal stability, DNA-binding ability, and transcriptional activity. The high efficacy of MANIO as an anticancer agent toward cancers harboring WT/mutp53 is further demonstrated in patient-derived cells and xenograft mouse models of colorectal cancer (CRC), with no signs of undesirable side effects. MANIO synergizes with conventional chemotherapeutic drugs, and in vitro and in vivo studies predict its adequate drug-likeness and pharmacokinetic properties for a clinical candidate. As a single agent or in combination, MANIO will advance anticancer-targeted therapy, particularly benefiting CRC patients harboring distinct p53 status.
Asunto(s)
Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Pirroles/farmacología , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/genética , Animales , Antineoplásicos/síntesis química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Doxorrubicina/farmacología , Descubrimiento de Drogas , Sinergismo Farmacológico , Femenino , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Unión Proteica , Pirroles/síntesis química , Tiazoles/síntesis química , Proteína p53 Supresora de Tumor/agonistas , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Combining low-dose chemotherapies is a strategy for designing less toxic and more potent childhood cancer treatments. We examined the effects of combining the novel thiosemicarbazones, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), or its analog, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), with the standard chemotherapies, celecoxib (CX), etoposide (ETO), or temozolomide (TMZ). These combinations were analyzed for synergism to inhibit proliferation of three pediatric tumor cell-types, namely osteosarcoma (Saos-2), medulloblastoma (Daoy) and neuroblastoma (SH-SY5Y). In terms of mechanistic dissection, this study discovered novel thiosemicarbazone targets not previously identified and which are important for considering possible drug combinations. In this case, DpC and Dp44mT caused: (1) up-regulation of a major protein target of CX, namely cyclooxygenase-2 (COX-2); (2) down-regulation of the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), which is known to affect TMZ resistance; (3) down-regulation of mismatch repair (MMR) proteins, MSH2 and MSH6, in Daoy and SH-SY5Y cells; and (4) down-regulation in all three cell-types of the MMR repair protein, MLH1, and also topoisomerase 2α (Topo2α), the latter of which is an ETO target. While thiosemicarbazones up-regulate the metastasis suppressor, NDRG1, in adult cancers, it is demonstrated herein for the first time that they induce NDRG1 in all three pediatric tumor cell-types, validating its role as a potential target. In fact, siRNA studies indicated that NDRG1 was responsible for MGMT down-regulation that may prevent TMZ resistance. Examining the effects of combining thiosemicarbazones with CX, ETO, or TMZ, the most promising synergism was obtained using CX. Of interest, a positive relationship was observed between NDRG1 expression of the cell-type and the synergistic activity observed in the combination of thiosemicarbazones and CX. These studies identify novel thiosemicarbazone targets relevant to childhood cancer combination chemotherapy.
RESUMEN
A newly established GM7 cell line was derived from the tumor tissue of a 65-year-old man surgically treated for a relapse of glioblastoma multiforme that occurred 10 months after first surgery following radiotherapy. GM7 cells exhibit spindle or glia-like morphology, and multinucleated giant cells are also present in the culture. The cells proliferate rapidly (PDT is about 18 h) and tend to grow in multilayer without contact inhibition. Using G-banding and SKY, the GM7 cell line was identified as near-triploid with a large number of structural and numerical abnormalities. Repeated karyotyping during long-term cultivation confirmed a chromosome number of 70+/-3 chromosomes per cell. Special attention was paid to the immunocytochemical analysis of protein markers in this cell line; GM7 cells showed strong positivity for CD133, vimentin, nestin, NF-160 and S-100 protein and weak positivity for GFAP and NSE, but were negative for synaptophysin. The most important features of the GM7 cell line are its stable phenotype CD133+/nestin+, which are accepted as stem cell markers in neural stem/progenitor cells, and especially unusual intracellular localization of the IF protein nestin, which was detected and repeatedly confirmed both in the cytoplasm and cell nucleus. For this reason, the new GM7 glioblastoma cell line represents an important model suitable not only for further studies on glioblastoma biology and cancer stem cells, but particularly for the detailed investigation of the role of nestin in transformed cells.
Asunto(s)
Antígenos CD/biosíntesis , Línea Celular Tumoral/fisiología , Línea Celular Tumoral/ultraestructura , Glioblastoma/metabolismo , Glicoproteínas/biosíntesis , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Antígeno AC133 , Anciano , Biomarcadores de Tumor/análisis , Western Blotting , Núcleo Celular/química , Núcleo Celular/metabolismo , Aberraciones Cromosómicas , Citoplasma/química , Citoplasma/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glioblastoma/genética , Glioblastoma/ultraestructura , Humanos , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Nestina , PéptidosRESUMEN
Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins (DDX39A, HMGA1, HMGA2, HOXC9 and PBX1) that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores Farmacológicos/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Isotretinoína/farmacología , Neuroblastoma/genética , Tretinoina/análogos & derivados , Tretinoina/farmacología , Adolescente , Bexaroteno/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Fenretinida/farmacología , Proteína HMGA1a/genética , Proteína HMGA1a/metabolismo , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Lactante , Recién Nacido , Masculino , Neoplasias del Sistema Nervioso/genética , Neoplasias del Sistema Nervioso/metabolismo , Neoplasias del Sistema Nervioso/patología , Neoplasias del Sistema Nervioso/cirugía , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuroblastoma/cirugía , Adhesión en Parafina , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismo , Fijación del Tejido , Adulto JovenRESUMEN
The survival rate for patients with high-risk neuroblastomas remains poor despite new improvements in available therapeutic modalities. A detailed understanding of the mechanisms underlying clinical responses to multimodal treatment is one of the important aspects that may provide precision in the prediction of a patient's clinical outcome. Our study was designed as a detailed comparative analysis of five selected proteins (DDX39A, HMGA1, HOXC9, NF1, and PBX1) in one cohort of patients using the same methodical approaches. These proteins were already reported separately as related to the resistance or sensitivity to retinoids and as useful prognostic markers of survival probability. In the cohort of 19 patients suffering from high-risk neuroblastomas, we analyzed initial immunohistochemistry samples obtained by diagnostic biopsy and post-induction samples taken after the end of induction therapy. The expression of DDX39A, HMGA1, HOXC9, and NF1 showed varied patterns with almost no differences between responders and non-responders. Nevertheless, we found very interesting results for PBX1: non-responders had significantly higher expression levels of this protein in the initial tumor samples when compared with responders; this expression pattern changed inversely in the post-induction samples, and this change was also statistically significant. Moreover, our results from survival analyses reveal the prognostic value of PBX1, NF1, and HOXC9 expression in neuroblastoma tissue. In addition to the prognostic importance of PBX1, NF1, and HOXC9 proteins, our results demonstrated that PBX1 could be used for the prediction of the clinical response to induction chemotherapy in patients suffering from high-risk neuroblastoma.
RESUMEN
TAp73 is a key tumor suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumors with impaired p53 signaling, like neuroblastoma, TAp73 activation represents an encouraging strategy, alternative to p53 activation, to suppress tumor growth and chemoresistance. In this work, we report a new TAp73-activating agent, the 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), with potent antitumor activity. Notably, LEM2 was able to release TAp73 from its interaction with both MDM2 and mutant p53, enhancing TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutant p53-expressing tumor cells. Importantly, LEM2 displayed potent antitumor activity against patient-derived neuroblastoma cells, consistent with an activation of the TAp73 pathway. Additionally, potent synergistic effects were obtained for the combination of LEM2 with doxorubicin and cisplatin in patient-derived neuroblastoma cells. Collectively, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against neuroblastoma.
Asunto(s)
Antineoplásicos/farmacología , Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Xantonas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HT29 , Humanos , Mutación , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Transducción de Señal/efectos de los fármacos , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Nestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines. METHODS: Using immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines. RESULTS: Nestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+. CONCLUSION: Our results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors.
Asunto(s)
Antígenos CD/genética , Glicoproteínas/genética , Proteínas de Filamentos Intermediarios/genética , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/genética , Osteosarcoma/genética , Péptidos/genética , Antígeno AC133 , Adolescente , Adulto , Antígenos CD/biosíntesis , Línea Celular Tumoral , Niño , Femenino , Técnica del Anticuerpo Fluorescente , Glicoproteínas/biosíntesis , Humanos , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/biosíntesis , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/patología , Proteínas del Tejido Nervioso/biosíntesis , Nestina , Osteosarcoma/patologíaRESUMEN
Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of ß-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of ß-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for ß-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.
Asunto(s)
Neoplasias Cerebelosas/patología , Cerebelo/patología , Meduloblastoma/patología , Adolescente , Adulto , Biomarcadores de Tumor/análisis , Niño , Preescolar , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunohistoquímica , Lactante , Masculino , Células Tumorales Cultivadas , Adulto JovenRESUMEN
BACKGROUND: Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors. METHODS: We examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. RESULTS: CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. CONCLUSION: Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors.
Asunto(s)
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Antígeno AC133 , Adolescente , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Niño , Preescolar , Estudios de Cohortes , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Nestina , Rabdomiosarcoma/genética , Células Madre/metabolismo , Adulto JovenRESUMEN
BACKGROUND: We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. METHODS: Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. RESULTS: Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. CONCLUSIONS: Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Regulación Neoplásica de la Expresión Génica , Inhibidores de la Lipooxigenasa/farmacología , Tretinoina/metabolismo , Neoplasias Encefálicas/patología , Ácidos Cafeicos/farmacología , Celecoxib , Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Humanos , Familia de Multigenes , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Estrés Oxidativo , Pirazoles/farmacología , Sulfonamidas/farmacologíaRESUMEN
We investigated the possible modulation by LOX/ COX inhibitors of all-trans retinoic acid (ATRA)-induced cell differentiation in two established neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor of cyclooxygenase-2, were chosen for this study. The effects of the combined treatment with ATRA and LOX/COX inhibitors on neuroblastoma cells were studied using cell morphology assessment, detection of differentiation markers by immunoblotting, measurement of proliferation activity, and cell cycle analysis and apoptosis detection by flow cytometry. The results clearly demonstrated the potential of caffeic acid to enhance ATRA-induced cell differentiation, especially in the SK-N-BE(2) cell line, whereas application of celecoxib alone or with ATRA led predominantly to cytotoxic effects in both cell lines. Moreover, the higher sensitivity of the SK-N-BE(2) cell line to combined treatment with ATRA and LOX/COX inhibitors suggests that cancer stem cells are a main target for this therapeutic approach. Nevertheless, further detailed study of the phenomenon of enhanced cell differentiation by expression profiling is needed.