RESUMEN
The protein p53 is one of the most important tumor suppressors, responding to a variety of stress signals. Mutations in p53 occur in about half of human cancer cases, and dysregulation of the p53 function by epigenetic modifiers and modifications is prevalent in a large proportion of the remainder. PRMT1 is the main enzyme responsible for the generation of asymmetric-dimethylarginine, whose upregulation or aberrant splicing has been observed in many types of malignancies. Here, we demonstrate that p53 function is regulated by PRMT1 in breast cancer cells. PRMT1 knockdown activated the p53 signal pathway and induced cell growth-arrest and senescence. PRMT1 could directly bind to p53 and inhibit the transcriptional activity of p53 in an enzymatically dependent manner, resulting in a decrease in the expression levels of several key downstream targets of the p53 pathway. We were able to detect p53 asymmetric-dimethylarginine signals in breast cancer cells and breast cancer tissues from patients, and the signals could be significantly weakened by silencing of PRMT1 with shRNA, or inhibiting PRMT1 activity with a specific inhibitor. Furthermore, PRMT1 inhibitors significantly impeded cell growth and promoted cellular senescence in breast cancer cells and primary tumor cells. These results indicate an important role of PRMT1 in the regulation of p53 function in breast tumorigenesis.
RESUMEN
PURPOSE: To investigate how necroptosis (ie, programmed necrosis) is involved in killing of non-small cell lung cancer (NSCLC) after ablative hypofractionated radiation therapy (HFRT). METHODS AND MATERIALS: Deoxyribonucleic acid damage, DNA repair, and the death form of NSCLC cells were assessed after radiation therapy. The overexpression and silencing of receptor-interacting protein kinases 3 (RIP3, a key protein involved activation of necroptosis)-stable NSCLC cell lines were successfully constructed. The form of cell death, the number and area of colonies, and the regulatory proteins of necroptosis were characterized after radiation therapy in vitro. Finally, NSCLC xenografts and patient specimens were used to examine involvement of necroptosis after ablative HFRT in vivo. RESULTS: Radiation therapy induced expected DNA damage and repair of NSCLC cell lines, but ablative HFRT at ≥10 Gy per fraction preferentially stimulated necroptosis in NSCLC cells and xenografts with high RIP3 expression, as characterized by induction and activation of RIP3 and mixed-lineage kinase domain-like protein and release of immune-activating chemokine high-mobility group box 1. In contrast, RNA interference of RIP3 attenuated ablative HFRT-induced necroptosis and activation of its regulatory proteins. Among central early-stage NSCLC patients receiving stereotactic body radiation therapy, high expression of RIP3 was associated with improved local control and progression-free survival (all P < .05). CONCLUSIONS: Ablative HFRT at ≥10 Gy per fraction enhances killing of NSCLC with high RIP3 expression via preferential stimulation of necroptosis. RIP3 may serve as a useful biomarker to predict favorable response to stereotactic body radiation therapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Necrosis , Hipofraccionamiento de la Dosis de Radiación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Muerte Celular/genética , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Quinasas Quinasa Quinasa PAM , Ratones , Ratones Desnudos , Necrosis/genética , Necrosis/patología , Fotograbar/métodos , ARN Interferente Pequeño , Tolerancia a Radiación , Radiocirugia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
BACKGROUND: E3 ubiquitin ligase Ring finger protein 180 (RNF180) has been identified as a novel tumor suppressor in gastric cancer and the methylated CpG site count of RNF180 DNA promoter can predict the prognosis for gastric cancer patients. OBJECTIVE: In the previous study, we demonstrated that methylated CpG site count of RNF180 DNA promoter was significantly associated with the survival of patients with gastric cancer using the bisulfite genomic sequencing (BGS) in the gastric cancer tissue with five clones per sample. It was so complicate for each patient underwent the BGS detection with clones. It is important to explore a simple, rapid and accurate method to detect methylated CpG site count to predicting the prognosis for gastric cancer patients. METHODS: At present study, we detected hypermethylated and hypomethylated CpG site count of RNF180 DNA promoter in samples of 480 gastric cancer patients by direct bisulfite sequencing. RESULTS: We found that patients who possessed seven or less hypermethylated CpG sites of RNF180 DNA promoter had much better survival (p= 0.008), which was similar to our previous research results by using the BGS with clones. With the multivariate survival analysis, we found that T stage, N stage and hypermethylated CpG site count of RNF180 DNA promoter were the independent predictors of prognosis for gastric cancer patients. CONCLUSIONS: hypermethylated CpG site count of RNF180 DNA promoter for evaluating the prognosis of gastric cancer was reasonable by using the direct bisulfite sequencing.
Asunto(s)
Metilación de ADN , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/genética , Ubiquitina-Proteína Ligasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Neoplasias Gástricas/mortalidad , Sulfitos , Tasa de Supervivencia , Adulto JovenRESUMEN
p204 overexpression in retinoblastoma (Rb)-/- mouse embryo fibroblasts or transfection of p204 mutated at both Rb-binding sites confer growth advantages, resulting in a significantly higher number of foci in a cell focus assay. To investigate the possibility that mutated p204 acquires malignant transformation capability, NIH3T3 cells were stably transfected with the expression vector pRcRSV204 double-mutant (p204dm) harboring both the C-terminal deletion up to amino acid 568 and the point mutation from glutamic acid to lysine at position 427, and analyzed for markers typical of cell immortalization and transformation. We detected a greater abundance of cell colonies in soft agar with p204dm-expressing cells than vector control cells. The p204dm-transfected cells also displayed two other characteristics associated with malignant transformation, i.e. growth under low-serum conditions and formation of tumors in athymic nude mice. Moreover, their telomerase activity was significantly higher than in the vector control cells. It would thus seem that p204, devoid of functional Rb-binding motifs, can become oncogenic.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína de Retinoblastoma/metabolismo , Células 3T3 , Agar , Animales , Sitios de Unión/fisiología , División Celular/efectos de los fármacos , División Celular/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas , Mutación Puntual , Eliminación de Secuencia , Telomerasa/análisis , Telomerasa/metabolismo , TransfecciónRESUMEN
Breast cancer is a disease in which cancer cells form in the tissues of the breast. The present study aimed to explore the effect of the flavonoid compound quercetin on the growth and apoptosis of human breast cancer cells. Varying concentrations (12.5, 25, 50, 100, 200 µM) of quercetin were applied to cultured MCF-7 human breast cancer cells for defined lengths of time. At 50 to 200 µM doses, quercetin significantly inhibited the proliferation of MCF-7 cells assessed by MTT colorimetry, in both dose- and time-dependent manners (P<0.05). The compound also increased apoptosis after 48 h of exposure (P<0.05). Furthermore, following quercetin treatment Bcl-2 expression decreased significantly while Bax expression increased significantly (P<0.05). In brief, quercetin inhibits cell growth and induces apoptosis in MCF-7 human breast cancer cells. The mechanisms behind these effects may stem from the downregulation of Bcl-2 protein expression and upregulation of Bax expression.