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1.
Int J Mol Sci ; 25(17)2024 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-39273404

RESUMEN

Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVß3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4-αVß3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of ß3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown ß3 Integrin upregulation. However, it is not known if astrocytes upregulate ß3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase ß3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated ß3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, ß3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.


Asunto(s)
Astrocitos , Adhesión Celular , Movimiento Celular , Gliosis , Astrocitos/metabolismo , Gliosis/metabolismo , Gliosis/patología , Animales , Antígenos Thy-1/metabolismo , Integrina alfaVbeta3/metabolismo , Inflamación/metabolismo , Inflamación/patología , Sindecano-4/metabolismo , Sindecano-4/genética , Ratones , Línea Celular , Humanos , Células Cultivadas , Transducción de Señal
2.
Int J Mol Sci ; 17(2): 260, 2016 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-26907262

RESUMEN

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4'-trimethoxy-2'-hydroxy-chalcone (CH1) and 3'-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/metabolismo , Caspasas/metabolismo , Chalconas/farmacología , Neoplasias Hepáticas/metabolismo , Animales , Apoptosis , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos
3.
Noncoding RNA ; 9(5)2023 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-37888205

RESUMEN

Breast cancer is currently the most diagnosed form of cancer and the leading cause of death by cancer among females worldwide. We described the family of long non-coding mitochondrial RNAs (ncmtRNAs), comprised of sense (SncmtRNA) and antisense (ASncmtRNA) members. Knockdown of ASncmtRNAs using antisense oligonucleotides (ASOs) induces proliferative arrest and apoptotic death of tumor cells, but not normal cells, from various tissue origins. In order to study the mechanisms underlying this selectivity, in this study we performed RNAseq in MDA-MB-231 breast cancer cells transfected with ASncmtRNA-specific ASO or control-ASO, or left untransfected. Bioinformatic analysis yielded several differentially expressed cell-cycle-related genes, from which we selected Aurora kinase A (AURKA) and topoisomerase IIα (TOP2A) for RT-qPCR and western blot validation in MDA-MB-231 and MCF7 breast cancer cells, as well as normal breast epithelial cells (HMEC). We observed no clear differences regarding mRNA levels but both proteins were downregulated in tumor cells and upregulated in normal cells. Since these proteins play a role in genomic integrity, this inverse effect of ASncmtRNA knockdown could account for tumor cell downfall whilst protecting normal cells, suggesting this approach could be used for genomic protection under cancer treatment regimens or other scenarios.

4.
Nanomedicine (Lond) ; 18(23): 1651-1668, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37929694

RESUMEN

Background: Elevated expression of CAV1 in breast cancer increases tumor progression. Extracellular vesicles (EVs) from CAV1-expressing MDA-MB-231 breast cancer cells contain Tenascin C (TNC), but the relevance of TNC remained to be defined. Methods: EVs were characterized by nanotracking analysis, microscopy and western blotting. The uptake of EVs by cells was studied using flow cytometry. The effects of EVs on breast cancer cells were tested in migration, invasion, colony formation and in vivo assays. Results: EVs were taken up by cells; however, only those containing TNC promoted invasiveness. In vivo, EVs lacking TNC ceased to promote tumor growth. Conclusion: CAV1 and TNC contained in breast cancer cell-derived EVs were identified as proteins that favor progression of breast cancer.


Caveolin-1 (CAV1) is a protein that in breast cancer increases with disease progression. Extracellular vesicles (EVs) from breast cancer cells with CAV1 also contain Tenascin C (TNC) protein, but the importance of TNC remained to be defined. EVs were identified by size, microscopy and protein analysis. The effects of EVs on breast cancer cells were studied using cells and experiments in animals. CAV1 expression promotes TNC inclusion into EVs, which increased the aggressiveness of recipient breast cancer cells. In animals, only EVs with TNC increased features associated with cancer spread, while EVs lacking TNC reduced tumor growth.


Asunto(s)
Neoplasias de la Mama , Caveolina 1 , Vesículas Extracelulares , Tenascina , Humanos , Línea Celular Tumoral , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caveolina 1/metabolismo , Vesículas Extracelulares/metabolismo , Tenascina/metabolismo , Animales , Ratones , Ratones SCID , Progresión de la Enfermedad
5.
Sci Rep ; 10(1): 343, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31941923

RESUMEN

During intercellular communication, cells release extracellular vesicles such as exosomes, which contain proteins, ncRNAs and mRNAs that can influence proliferation and/or trigger apoptosis in recipient cells, and have been proposed to play an essential role in promoting invasion of tumor cells and in the preparation of metastatic niches. Our group proposed the antisense non-coding mitochondrial RNA (ASncmtRNA) as a new target for cancer therapy. ASncmtRNA knockdown using an antisense oligonucleotide (ASO-1537S) causes massive death of tumor cells but not normal cells and strongly reduces metastasis in mice. In this work, we report that exosomes derived from ASO-1537S-treated MDA-MB-231 breast cancer cells (Exo-1537S) inhibits tumorigenesis of recipient cells, in contrast to exosomes derived from control-ASO-treated cells (Exo-C) which, in contrast, enhance these properties. Furthermore, an in vivo murine peritoneal carcinomatosis model showed that Exo-1537S injection reduced tumorigenicity compared to controls. Proteomic analysis revealed the presence of Lactadherin and VE-Cadherin in exosomes derived from untreated cells (Exo-WT) and Exo-C but not in Exo-1537S, and the latter displayed enrichment of proteasomal subunits. These results suggest a role for these proteins in modulation of tumorigenic properties of exosome-recipient cells. Our results shed light on the mechanisms through which ASncmtRNA knockdown affects the preparation of breast cancer metastatic niches in a peritoneal carcinomatosis model.


Asunto(s)
Exosomas/metabolismo , Mitocondrias/genética , ARN no Traducido/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Superficie/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Proteínas de la Leche/metabolismo , Oligorribonucleótidos Antisentido/metabolismo , Oligorribonucleótidos Antisentido/farmacología , ARN no Traducido/antagonistas & inhibidores , ARN no Traducido/genética , Trasplante Heterólogo
6.
Aging (Albany NY) ; 11(1): 33-47, 2018 12 30.
Artículo en Inglés | MEDLINE | ID: mdl-30595560

RESUMEN

Human and mouse cells display a differential expression pattern of a family of mitochondrial noncoding RNAs (ncmtRNAs), according to proliferative status. Normal proliferating and cancer cells express a sense ncmtRNA (SncmtRNA), which seems to be required for cell proliferation, and two antisense transcripts referred to as ASncmtRNA-1 and -2. Remarkably however, the ASncmtRNAs are downregulated in human and mouse cancer cells, including HeLa and SiHa cells, transformed with HPV-18 and HPV-16, respectively. HPV E2 protein is considered a tumor suppressor in the context of high-risk HPV-induced transformation and therefore, to explore the mechanisms involved in the downregulation of ASncmtRNAs during tumorigenesis, we studied human foreskin keratinocytes (HFK) transduced with lentiviral-encoded HPV-18 E2. Transduced cells displayed a significantly extended replicative lifespan of up to 23 population doublings, compared to 8 in control cells, together with downregulation of the ASncmtRNAs. At 26 population doublings, cells transduced with E2 were arrested at G2/M, together with downregulation of E2 and SncmtRNA and upregulation of ASncmtRNA-2. Our results suggest a role for high-risk HPV E2 protein in cellular immortalization. Additionally, we propose a new cellular phenotype according to the expression of the SncmtRNA and the ASncmtRNAs.


Asunto(s)
Senescencia Celular/fisiología , Papillomavirus Humano 18/metabolismo , Queratinocitos/fisiología , Proteínas Oncogénicas Virales/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mitocondrial/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Regulación hacia Abajo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , ARN Mitocondrial/genética , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo
7.
Oncotarget ; 8(27): 43692-43708, 2017 07 04.
Artículo en Inglés | MEDLINE | ID: mdl-28620146

RESUMEN

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma.


Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , ARN sin Sentido , ARN no Traducido , ARN , Animales , Apoptosis/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Ratones , Metástasis de la Neoplasia , ARN Mitocondrial , Ensayos Antitumor por Modelo de Xenoinjerto
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