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1.
J Cell Biochem ; 125(2): e30517, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38224178

RESUMEN

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.


Asunto(s)
Neoplasias Colorrectales , Humanos , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Neoplasias Colorrectales/genética , Recurrencia Local de Neoplasia , Células HT29 , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Apoptosis , Autofagia , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética
2.
Biochem Biophys Res Commun ; 517(2): 324-329, 2019 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-31353083

RESUMEN

Induced pluripotent stem cells (iPSC) have a great potential, but their clinical application depends on finding strategies to abolish their tumorigenic potential. The use of Oct4, Sox2, Klf4, c-Myc and Nanog to generate iPSC demonstrated the already known importance of these genes to maintain stemness. Therefore, the presence of these genes is responsible for iPSC-derived teratomas. Similar to iPSC, P19 teratocarcinoma cell line also has characteristics of embryonic carcinoma cells and the ability to differentiate into many cell types. We separately silenced the transcription factors Oct4, Sox2, Klf4, c-Myc and Nanog in P19 cells and measured the impact of this silencing in vivo. All silenced cells generated tumors when injected in immunosuppressed mice, but silencing of Oct4, Sox2 and Klf4 generated mainly teratomas with mesoderm tissue. Our results suggest that downregulation of these transcription factors is not enough to avoid the formation of teratomas, but their silencing affect their differentiation potential.


Asunto(s)
Silenciador del Gen , Teratoma/genética , Factores de Transcripción/genética , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular , Proliferación Celular , Femenino , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción SOXB1/genética , Teratoma/patología
3.
Mol Neurobiol ; 55(5): 4185-4194, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-28612256

RESUMEN

Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5 µM) and DOX (1 and 10 nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35 days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7 days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood-brain barrier permeability.


Asunto(s)
Neoplasias Encefálicas/patología , Doxorrubicina/farmacología , Glioblastoma/patología , Temozolomida/farmacología , Recuento de Células , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología
4.
Mol Neurobiol ; 54(10): 7610-7619, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27832522

RESUMEN

Neuroblastoma (NB) is the most common extracranial solid childhood tumor accounting for around 15% of pediatric cancer deaths and most probably originates from a failure in the development of embryonic neural crest cells. Retinoids can inhibit the proliferation and stimulate differentiation of NB cells. In addition, epigenetic events involving changes in chromatin structure and DNA methylation can mediate the effects of retinoids; hence, the scope of this study is to investigate the use of retinoids and epigenetic drugs in NB cell lines. Here, we demonstrate that the combination of retinoid all trans-retinoic acid (ATRA) with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase is more effective in impairing the proliferation of human SH-SY5Y and SK-N-BE(2) NB cells than any drug given alone. Treatments also induced differential changes on the messenger RNA (mRNA) expression of retinoid receptor subtypes and reduced the protein content of c-Myc, the neuronal markers NeuN and ß-3 tubulin, and the oncoprotein Bmi1. These results suggest that the combination of retinoids with epigenetic modulators is more effective in reducing NB growth than treatment with single drugs.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Neuroblastoma/metabolismo , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Epigénesis Genética/fisiología , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Esteroides/administración & dosificación , Tretinoina/administración & dosificación , Tretinoina/análogos & derivados
5.
Endocr Relat Cancer ; 23(3): 135-46, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26825960

RESUMEN

Type 3 deiodinase (DIO3, D3) is reactivated in human neoplasias. Increased D3 levels in papillary thyroid carcinoma (PTC) have been associated with tumor size and metastatic disease. The objective of this study is to investigate the signaling pathways involved in DIO3 upregulation in PTC. Experiments were performed in human PTC cell lines (K1 and TPC-1 cells) or tumor samples. DIO3 mRNA and activity were evaluated by real-time PCR and ion-exchange column chromatography respectively. Western blot analysis was used to determine the levels of D3 protein. DIO3 gene silencing was performed via siRNA transfection. DIO3 mRNA levels and activity were readily detected in K1 (BRAF(V6) (0) (0E)) and, at lower levels, in TPC-1 (RET/PTC1) cells (P<0.007 and P=0.02 respectively). Similarly, DIO3 mRNA levels were higher in PTC samples harboring the BRAF(V600E) mutation as compared with those with RET/PTC1 rearrangement or negative for these mutations (P<0.001). Specific inhibition of BRAF oncogene (PLX4032, 3 µM), MEK (U0126, 10-20 µM) or p38 (SB203580, 10-20 µM) signaling was associated with decreases in DIO3 expression in K1 and TPC-1 cells. Additionally, the blockage of the sonic hedgehog (SHH) pathway by cyclopamine (10  µM) resulted in markedly decreases in DIO3 mRNA levels. Interestingly, siRNA-mediated DIO3 silencing induced decreases on cyclin D1 expression and partial G1 phase cell cycle arrest, thereby downregulating cell proliferation. In conclusion, sustained activation of the MAPK and SHH pathways modulate the levels of DIO3 expression in PTC. Importantly, DIO3 silencing was associated with decreases in cell proliferation, thus suggesting a D3 role in tumor growth and aggressiveness.


Asunto(s)
Carcinoma/metabolismo , Proteínas Hedgehog/metabolismo , Yoduro Peroxidasa/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias de la Tiroides/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Carcinoma/genética , Carcinoma Papilar , Línea Celular Tumoral , Proliferación Celular , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Yoduro Peroxidasa/genética , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Mutación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores
6.
Oncotarget ; 7(23): 34860-80, 2016 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-27145455

RESUMEN

Ewing sarcoma (ES) is a highly aggressive pediatric cancer that may arise from neuronal precursors. Neurotrophins stimulate neuronal devlopment and plasticity. Here, we found that neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as their receptors (TrkA and TrkB, respectively) are expressed in ES tumors. Treatment with TrkA (GW-441756) or TrkB (Ana-12) selective inhibitors decreased ES cell proliferation, and the effect was increased when the two inhibitors were combined. ES cells treated with a pan-Trk inhibitor, K252a, showed changes in morphology, reduced levels of ß-III tubulin, and decreased mRNA expression of NGF, BDNF, TrkA and TrkB. Furthermore, combining K252a with subeffective doses of cytotoxic chemotherapeutic drugs resulted in a decrease in ES cell proliferation and colony formation, even in chemoresistant cells. These results indicate that Trk inhibition may be an emerging approach for the treatment of ES.


Asunto(s)
Antineoplásicos/farmacología , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Glicoproteínas de Membrana/antagonistas & inhibidores , Factor de Crecimiento Nervioso/biosíntesis , Receptor trkA/antagonistas & inhibidores , Receptor trkB/antagonistas & inhibidores , Sarcoma de Ewing/tratamiento farmacológico , Azepinas/farmacología , Benzamidas/farmacología , Factor Neurotrófico Derivado del Encéfalo/genética , Carbazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Humanos , Alcaloides Indólicos/farmacología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Factor de Crecimiento Nervioso/genética , ARN Mensajero/biosíntesis , Receptor trkA/biosíntesis , Receptor trkA/genética , Receptor trkB/biosíntesis , Receptor trkB/genética , Sarcoma de Ewing/patología , Tubulina (Proteína)/metabolismo , Vincristina/farmacología
7.
PLoS One ; 10(3): e0120538, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25756665

RESUMEN

Glucocorticoids and immunosuppressive drugs are commonly used to treat inflammatory disorders, such as inflammatory bowel disease (IBD), and despite a few improvements, the remission of IBD is still difficult to maintain. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have emerged as regulators of the immune response, and their viability and activation of their migratory properties are essential for successful cell therapy. However, little is known about the effects of immunosuppressant drugs used in IBD treatment on MSC behavior. The aim of this study was to evaluate MSC viability, nuclear morphometry, cell polarity, F-actin and focal adhesion kinase (FAK) distribution, and cell migratory properties in the presence of the immunosuppressive drugs azathioprine (AZA) and dexamethasone (DEX). After an initial characterization, MSCs were treated with DEX (10 µM) or AZA (1 µM) for 24 hrs or 7 days. Neither drug had an effect on cell viability or nuclear morphometry. However, AZA treatment induced a more elongated cell shape, while DEX was associated with a more rounded cell shape (P < 0.05) with a higher presence of ventral actin stress fibers (P < 0.05) and a decrease in protrusion stability. After 7 days of treatment, AZA improved the cell spatial trajectory (ST) and increased the migration speed (24.35%, P < 0.05, n = 4), while DEX impaired ST and migration speed after 24 hrs and 7 days of treatment (-28.69% and -25.37%, respectively; P < 0.05, n = 4). In conclusion, our data suggest that these immunosuppressive drugs each affect MSC morphology and migratory capacity differently, possibly impacting the success of cell therapy.


Asunto(s)
Azatioprina/farmacología , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Inmunosupresores/farmacología , Células Madre Mesenquimatosas/fisiología , Forma de la Célula/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Citoesqueleto/metabolismo , Evaluación Preclínica de Medicamentos , Quinasa 1 de Adhesión Focal/metabolismo , Humanos
8.
J Mol Neurosci ; 54(1): 27-40, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24464841

RESUMEN

Epigenetic alterations have been increasingly implicated in glioblastoma (GBM) pathogenesis, and epigenetic modulators including histone deacetylase inhibitors (HDACis) have been investigated as candidate therapies. GBMs are proposed to contain a subpopulation of glioblastoma stem cells (GSCs) that sustain tumor progression and therapeutic resistance and can form tumorspheres in culture. Here, we investigate the effects of the HDACi trichostatin A (TSA) in U87 GBM cultures and tumorsphere-derived cells. Using approaches that include a novel method to measure tumorsphere sizes and the area covered by spheres in GBM cultures, as well as a nuclear morphometric analysis, we show that TSA reduced proliferation and colony sizes, led to G2/M arrest, induced alterations in nuclear morphology consistent with cell senescence, and increased the protein content of GFAP, but did not affect migration, in cultured human U87 GBM cells. In cells expanded in tumorsphere assays, TSA reduced sphere formation and induced neuron-like morphological changes. The expression of stemness markers in these cells was detected by reverse transcriptase polymerase chain reaction. These findings indicate that HDACis can inhibit proliferation, survival, and tumorsphere formation, and promote differentiation of U87 GBM cells, providing further evidence for the development of HDACis as potential therapeutics against GBM.


Asunto(s)
Antineoplásicos/farmacología , Glioblastoma/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Células Tumorales Cultivadas
9.
World J Gastroenterol ; 20(48): 18228-39, 2014 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-25561790

RESUMEN

AIM: To investigate the therapeutic effects of mesenchymal stem cells (MSCs) transplanted intraperitoneally and intravenously in a murine model of colitis. METHODS: MSCs were isolated from C57BL/6 mouse adipose tissue. MSC cultures were analyzed according to morphology, cellular differentiation potential, and surface molecular markers. Experimental acute colitis was induced in C57BL/6 mice by oral administration of 2% dextran sulfate sodium (DSS) in drinking water ad libitum from days 0 to 7. Colitis mice were treated with 1 × 10(6) MSCs via intraperitoneal or intravenous injection on days 2 and 5. The disease activity index was determined daily based on the following parameters: weight loss, stool consistency and presence of blood in the feces and anus. To compare morphological and functional differences in tissue regeneration between different MSC injection modalities, mice were euthanized on day 8, and their colons were examined for length, weight, and histopathological changes. Inflammatory responses were determined by measuring the levels of different serum cytokines using a CBA Th1/Th2/Th17 kit. Apoptotic rates were evaluated by terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling assay. RESULTS: Intravenous infusion of MSCs was more effective than intraperitoneal treatment (P < 0.001) in reducing the clinical and histopathologic severity of colitis, which includes weight loss, diarrhea and inflammation. An histological evaluation demonstrated decreased colonic inflammation based on reduced crypt loss and reduced infiltration of inflammatory cells. This therapeutic effect was most likely mediated by the down-regulation of pro-inflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)]; and by the up-regulation of anti-inflammatory cytokines (IL-10 and IL-4). Intravenous transplantation also induced high levels of IFN that lead to activation of the immunosuppressive activity of the MSCs, which did not occur with intraperitoneal transplantation (P = 0.006). An increase in apoptotic T cells was observed after intravenous, but not intraperitoneal, MSC infusion, suggesting that MSCs can induce apoptosis in resistant T cells in colonic inflammation (P = 0.027). CONCLUSION: Our results demonstrate that intravenous treatment is a superior method for reducing colon inflammation compared with intraperitoneal therapy.


Asunto(s)
Colitis/cirugía , Colon/fisiopatología , Trasplante de Células Madre Mesenquimatosas/métodos , Regeneración , Enfermedad Aguda , Animales , Apoptosis , Biomarcadores/sangre , Células Cultivadas , Colitis/sangre , Colitis/inducido químicamente , Colitis/patología , Colitis/fisiopatología , Colon/metabolismo , Colon/patología , Citocinas/sangre , Sulfato de Dextran , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Infusiones Intravenosas , Infusiones Parenterales , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Fenotipo , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Tiempo
10.
Genomic Med ; 1(1-2): 57-63, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18923929

RESUMEN

Protein p53 is the tumor suppressor involved in cell cycle control and apoptosis. There are several polymorphisms reported for p53 which can affect important regions involved in protein tumor suppressor activity. Amongst the polymorphisms described, R213R and 13949 g-->a are rarely studied, with an estimate frequency not yet available for the Brazilian population. The purpose of this study was to investigate the genotype and allele frequencies and associations of these polymorphisms in a group of patients with altered esophageal tissue from South Brazil and compare with the frequency observed for a control population. A total of 35 patients for R213R and 45 for 13494 g-->a polymorphisms analysis with gastroesophageal reflux disease (GERD) symptoms diagnosed by upper digestive endoscopy and confirmed by biopsy were studied. For both groups, 100 controls were used for comparison. Loss of heterozygosity (LOH) was also analyzed for a selected group of patients where normal and affected tissue was available. There was one patient with Barrett's Esophagus (BE) showing LOH for R213R out of two heterozygous samples analyzed and two patients (esophagitis and BE) for 13494 g-->a polymorphism. We also aimed to build a haplotype for both polymorphisms collectively analyzed with R27P polymorphism, previously reported by our group. There were no significant differences in allele and genotype distribution between patients and controls. Although using esophagitis, intestinal metaplasia of the cardia and BE samples, all non-neoplastic lesions, we can conclude that these sites do not represent genetic susceptibility markers for the development and early progression of GERD to BE and esophageal cancer. Additional studies are required in order to investigate other determiners of early premalignant lesions known to predispose to esophageal cancer.

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