RESUMEN
Non-alcoholic steatohepatitis (NASH) has pathological characteristics similar to those of alcoholic hepatitis, despite the absence of a drinking history. The greatest threat associated with NASH is its progression to cirrhosis and hepatocellular carcinoma. The pathophysiology of NASH is not fully understood to date. In this study, we investigated the pathophysiology of NASH from the perspective of glycolysis and the Warburg effect, with a particular focus on microRNA regulation in liver-specific macrophages, also known as Kupffer cells. We established NASH rat and mouse models and evaluated various parameters including the liver-to-body weight ratio, blood indexes, and histopathology. A quantitative phosphoproteomic analysis of the NASH rat model livers revealed the activation of glycolysis. Western blotting and immunohistochemistry results indicated that the expression of pyruvate kinase muscle 2 (PKM2), a rate-limiting enzyme of glycolysis, was upregulated in the liver tissues of both NASH models. Moreover, increases in PKM2 and p-PKM2 were observed in the early phase of NASH. These observations were partially induced by the downregulation of microRNA122-5p (miR-122-5p) and occurred particularly in the Kupffer cells. Our results suggest that the activation of glycolysis in Kupffer cells during NASH was partially induced by the upregulation of PKM2 via miR-122-5p suppression.
Asunto(s)
Neoplasias Hepáticas , MicroARNs , Enfermedad del Hígado Graso no Alcohólico , Piruvato Quinasa/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Glucólisis , Macrófagos del Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Músculos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Piruvato Quinasa/genética , RatasRESUMEN
Cancer-related microRNAs (miRNAs) are emerging as promising and noninvasive biomarkers for colorectal cancer (CRC). This study aimed to investigate the usefulness of postoperative changes in plasma miR21-5p levels for recurrence and progressive disease (PD) after surgical resection. This study was a prospective study of 103 CRC patients who underwent surgical resection. Self-paired plasma samples collected pre-operation (Pre), 7 days post-operation (POD7), 1 month post-operation (POM1), and 6 months post-operation (POM6) were analyzed. The miRNA levels were evaluated by quantitative reverse transcription PCR. Among the enrolled patients, ten cases (9.7%) of postoperative recurrence and six cases (5.8%) of postoperative PD occurred at POM6. In the recurrence and PD group, plasma miR21-5p levels significantly increased (POM1: P < .01, POM6: P < .01, respectively). The area under the curve (AUC) value for postoperative changes in plasma miR21-5p levels at POM1 and POM6 to discriminate recurrence and PD were 0.675 and 0.715, respectively. Combined analysis with postoperative carcinoembryonic antigen (CEA) level in discriminating recurrence and PD increased AUC values (POM1: 0.715 and POM6: 0.789). Furthermore, multivariate analysis for recurrence and PD after surgical resection showed that postoperative changes in the plasma miR21-5p level at POM1 and POM6 were independent prognostic factors (POM1: P = .03, POM6: P < .01). The postoperative changes in plasma miR21-5p level could be a useful noninvasive biomarker for monitoring and predicting recurrence and PD after surgical resection of CRC patients. Furthermore, plasma miR21-5p can predict recurrence and PD after surgical resection.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/sangre , MicroARNs/sangre , Recurrencia Local de Neoplasia/sangre , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Antígeno Carcinoembrionario/sangre , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/cirugía , Diagnóstico Diferencial , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/cirugía , Periodo Posoperatorio , Estudios Prospectivos , Factores de TiempoRESUMEN
To understand the role of RAS-signaling networks in the pathogenesis of renal cell carcisnoma, we clarified the relationship between miR-143 and RAS. The expression of miR-143 was extremely downregulated in tumor tissues from renal cell carcinoma patients compared with that in the adjacent normal tissues and Caki-1 cells. We developed a synthetic miR-143#12, and we found that the ectopic expression of it inhibited cell growth with autophagy in Caki-1 cells. Also, the expression level of c-Myc was markedly decreased, resulting in the perturbation of cancer-specific energy metabolism by negatively modulating the expression of GLUT1 and the PTBP1/PKMs axis. A partial metabolic shift from glycolysis to oxidative phosphorylation induced autophagy through increasing the intracellular level of reactive oxygen species (ROS). In an in vivo study, the potent anti-tumor activity of polyion complex (PIC)-loaded miR-143#12 (miR-143#12/PIC) was shown by systemic administration of it to Caki-1 cell-xenografted mice. Higher levels of miR-143 were found in both blood and tumor tissues after the systemic administration with miR-143#12/PIC compared to those with lipoplexes in the xenografted mice. These findings indicated that this synthetic miR-143#12 induced a marked growth inhibition by impairing K-RAS-signaling networks in vitro and in vivo.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/terapia , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Autofagia/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Transportador de Glucosa de Tipo 1/genética , Glucólisis/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Ratones , MicroARNs/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Proteína de Unión al Tracto de Polipirimidina/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
microRNA-143(miR-143) is a well-known tumor suppressive microRNA that exhibits anti-tumoral function by targeting KRAS signaling pathways in various malignancies. We hypothesized that miR-143 suppresses breast cancer progression by targeting KRAS and its effector molecules. We further hypothesized that high expression of miR-143 is associated with a favorable tumor immune microenvironment of estrogen receptor (ER)-positive breast cancer patients which result in improved survival. Two major publicly available breast cancer cohorts; The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) were used. The miR-143 high expression group was associated with increased infiltration of anti-cancer immune cells and decreased pro-cancer immune cells, as well as enrichment of the genes relating to T helper (Th1) cells resulting in improved overall survival (OS) in ER-positive breast cancer patients. To the best of our knowledge, this is the first study to demonstrate that high expression of miR-143 in cancer cells associates with a favorable tumor immune microenvironment, upregulation of anti-cancer immune cells, and suppression of the pro-cancer immune cells, associating with better survival of the breast cancer patients.
Asunto(s)
Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal , Microambiente Tumoral/genética , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/genética , Biomarcadores de Tumor , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Therapy based on targeted inhibition of BCR-ABL tyrosine kinase has greatly improved the prognosis for patients with Philadelphia chromosome (Ph)-positive leukemia and tyrosine kinase inhibitors (TKI) have become standard therapy. However, some patients acquire resistance to TKI that is frequently associated with point mutations in BCR-ABL. We previously reported that a medium-chain fatty-acid derivative AIC-47 induced transcriptional suppression of BCR-ABL and perturbation of the Warburg effect, leading to growth inhibition in Ph-positive leukemia cells. Herein, we showed that AIC-47 had anti-leukemic effects in either wild type (WT)- or mutated-BCR-ABL-harboring cells. AIC-47 suppressed transcription of BCR-ABL gene regardless of the mutation through downregulation of transcriptional activator, c-Myc. Reprogramming of the metabolic pathway has been reported to be associated with resistance to anti-cancer drugs; however, we found that a point mutation of BCR-ABL was independent of the profile of pyruvate kinase muscle (PKM) isoform expression. Even in T315I-mutated cells, AIC-47 induced switching of the expression profile of PKM isoforms from PKM2 to PKM1, suggesting that AIC-47 disrupted the Warburg effect. In a leukemic mouse model, AIC-47 greatly suppressed the increase in BCR-ABL mRNA level and improved hepatosplenomegaly regardless of the BCR-ABL mutation. Notably, the improvement of splenomegaly by AIC-47 was remarkable and might be equal to or greater than that of TKI. These findings suggest that AIC-47 might be a promising agent for overcoming the resistance of Ph-positive leukemia to therapy.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Ácidos Grasos/farmacología , Proteínas de Fusión bcr-abl/genética , Compuestos Heterocíclicos con 1 Anillo/farmacología , Cetonas/farmacología , Leucemia/tratamiento farmacológico , Mutación Puntual/genética , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Leucemia/genética , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
It has been well established that microRNA (miR)-143 is downregulated in human bladder cancer (BC). Recent precision medicine has shown that mutations in BC are frequently observed in FGFR3, RAS and PIK3CA genes, all of which correlate with RAS signaling networks. We have previously shown that miR-143 suppresses cell growth by inhibiting RAS signaling networks in several cancers including BC. In the present study, we showed that synthetic miR-143 negatively regulated the RNA-binding protein Musashi-2 (MSI2) in BC cell lines. MSI2 is an RNA-binding protein that regulates the stability of certain mRNAs and their translation by binding to the target sequences of the mRNAs. Of note, the present study clarified that MSI2 positively regulated KRAS expression through directly binding to the target sequence of KRAS mRNA and promoting its translation, thus contributing to the maintenance of KRAS expression. Thus, miR-143 silenced KRAS and MSI2, which further downregulated KRAS expression through perturbation of the MSI2/KRAS cascade.
Asunto(s)
MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas de Unión al ARN/genética , Neoplasias de la Vejiga Urinaria/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas de Unión al ARN/metabolismo , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Macrophages are polarized into functional classically activated and alternatively activated (M2) phenotypes depending on their microenvironment, and these cells play an important role in the immune system. M2-like polarization of tumor-associated macrophages (TAMs) is activated by various secretions from cancer cells; however, the interaction between cancer cells and TAMs is not well understood. Recent studies showed that cancer cell-derived extracellular vesicles (EVs) contribute to tumor development and modulation of the tumor microenvironment. In the current study, we investigated colorectal cancer-derived EVs containing miR-145 with respect to the polarization of TAMs. Colorectal cancer cells positively secreted miR-145 via EVs, which were taken up by macrophage-like cells. Interestingly, colorectal cancer-derived EVs polarized macrophage-like cells into the M2-like phenotype through the downregulation of histone deacetylase 11 An in vivo study showed that EV-treated macrophages caused significant enlargement of the tumor volumes. These findings suggest that colorectal cancer cells use miR-145 within EVs to efficiently modulate M2-like macrophage polarization and tumor progression.
Asunto(s)
Neoplasias Colorrectales/inmunología , Vesículas Extracelulares/fisiología , Macrófagos/inmunología , MicroARNs/metabolismo , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Regulación hacia Abajo , Vesículas Extracelulares/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Activación de Macrófagos , Ratones , Ratones Desnudos , MicroARNs/genética , Fenotipo , Microambiente Tumoral/genéticaRESUMEN
Gastric cancer (GC) is one of the most common cancers worldwide. In the clinical setting, the identification of HER2 overexpression in GC was a significant finding, as trastuzumab, an anti-HER2 drug, provides a survival advantage to HER2-positive GC patients. In HER2-postive GC, the dysregulation of PI3K/AKT and MAPK/ERK signaling pathways has been reported, and inhibition of these pathways is an important therapeutic strategy. MiR-143 is known to act as a tumor suppressor in several cancers, such as bladder cancer, breast cancer, colorectal cancer, and gastric cancer. In the current study, we developed a novel chemically-modified miR-143 and explored the functions of this synthetic miR-143 (syn-miR-143) in HER2-positive gastric cancer. The expression level of miR-143 was down-regulated in GC cell lines, including HER2-positive GC cell lines, MKN7, and KATO-III. The ectopic expression of miR-143 in those cell lines suppressed cell growth through systemic silencing of KRAS and its effector signaling molecules, AKT and ERK. Furthermore, syn-miR-143 indirectly down-regulated the expression of HER2, an upstream molecule of KRAS, through silencing DEAD/H-box RNA helicase 6 (DDX6), RNA helicase, which enhanced HER2 protein expression at the translational step in HER2-positive GC cells. These findings suggested that syn-miR-143 acted as a tumor suppressor through the impairment of KRAS networks including the DDX6.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Animales , Antagomirs/metabolismo , Apoptosis/genética , Secuencia de Bases , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones Desnudos , MicroARNs/genética , Modelos Biológicos , Transducción de Señal , Regulación hacia Arriba/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC50 : 1.32 nmol L-1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , MicroARNs/administración & dosificación , MicroARNs/síntesis química , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Bencimidazoles/administración & dosificación , Bencimidazoles/farmacología , Benzotiazoles/administración & dosificación , Benzotiazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Sinergismo Farmacológico , Flavonoides/administración & dosificación , Flavonoides/farmacología , Células HT29 , Humanos , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/química , MicroARNs/farmacología , Mutación , Trasplante de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Human DEAD-box RNA helicase gene DDX6 was cloned from B-cell lymphoma cell line RC-K8. Previously, we reported that DDX6 acts as oncogene in several cancers such as colorectal cancer and hepatocellular carcinoma. However, the detailed mechanism of DDX6 action in carcinogenesis is largely unknown. In this study, we examined the functions of DDX6 in clinical gastric cancer (GC) samples and GC cells. DDX6 protein expression levels of cancer samples were higher than those of the adjacent normal tissues in 25 clinical GC samples (median value: 1.4 times higher). Also, the results of an RNA immunoprecipitation-assay (RIP-assay) showed that DDX6 associated with c-Myc mRNA. Moreover, enforced overexpression of DDX6 promoted both mRNA and protein expression of c-Myc in GC cells. On the other hand, the gene silencing of DDX6 induced growth suppression through down-regulation of c-Myc in GC cells grown in either two or three dimensions. Furthermore, c-Myc mRNA expression levels of cancer samples were higher than those of the adjacent normal tissues in DDX6 up-regulated-GC clinical samples. Our findings in this study suggested that DDX6 acted as oncogene in GC cells through promotion of c-Myc expression by association with the mRNA of c-Myc.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Gástricas/metabolismo , Regulación hacia Arriba , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Proliferación Celular , ARN Helicasas DEAD-box/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias Gástricas/genéticaRESUMEN
Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.
Asunto(s)
Neoplasias del Colon/metabolismo , Metabolismo Energético , Piruvato Quinasa/genética , Empalme del ARN/genética , Factores de Empalme Serina-Arginina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Autofagia , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/patología , Neoplasias del Colon/ultraestructura , Femenino , Silenciador del Gen , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones Desnudos , Persona de Mediana Edad , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Piruvato Quinasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The human DEAD/H-box RNA helicase DDX6 (RCK/p54) is a protein encoded by the fusion gene from the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma cell line RC-K8. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, and ribosome assembly. However, details of the regulatory mechanism governing DDX6 and the functions of DDX6 are largely unknown. Previously, we reported that DDX6 is overexpressed in most malignant cell lines and clinical colorectal tumor samples and that DDX6 positively contributes to the pathogenesis of various cancers. In the current study, we aimed at revealing the function of DDX6 in HER2 and FGFR2 related human gastric cancer (GC) by using clinical samples and GC cell lines. DDX6 protein was overexpressed in about 60% of the clinical samples; HER2, in 35%; and FGFR2, in 30%, (n = 20). Interestingly, the DDX6 protein was overexpressed in all HER2-positive samples (n = 7), and in 83% (5 of 6) of the FGFR2-positive samples, which could reflect the contribution of DDX6 to the expression of HER2 and FGFR2. In the GC cell line MKN7, which has HER2 amplification, the knockdown of DDX6 by siR-DDX6 led to the decreased expression of the HER2 protein. On the other hand, the knockdown of HER2 did not influence the DDX6 expression. Similar results were also obtained for the KATO-III and HSC39 cell lines having amplified FGFR2 expression. The increased expression of DDX6 induced a significantly increased expression of the HER2 protein without increasing the mRNA expression. The results of an RNP Immunoprecipitation (RIP)-assay using GC cells indicated that the DDX6 protein acted as an RNA-binding protein for HER2 and FGFR2 mRNAs and positively regulated their post-transcriptional processes. These findings demonstrated that DDX6 was an upstream molecule that positively regulated the expression of HER2 and FGFR2 at the post-transcriptional step in GC cells.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Neoplasias Gástricas/metabolismo , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias Gástricas/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Pyruvate kinase is known as the glycolytic enzyme catalyzing the final step in glycolysis. In mammals, two different forms of it exist, i.e., pyruvate kinase M1/2 (PKM) and pyruvate kinase L/R (PKLR). Also, PKM has two isoforms, i.e., PKM1 and PKM2. These genes have tissue-specific distribution. Namely, PKM1 is distributed in high-energy-demanding organs, such as brain and muscle. Also, PKM2 is distributed in various other organs, such as the colon. On the other hand, PKLR is distributed in liver and red blood cells (RBCs). Interestingly, PKM2 has been recognized as one of the essential genes for the cancer-specific energy metabolism termed the “Warburg effect”. However, the mechanism(s) underlying this fact have remained largely unclear. Recently, we found that some organ-specific microRNAs (miRNAs, MIR) regulate PKM isoform expression through direct targeting of polypyrimidine tract binding protein 1 (PTBP1), which is the splicer responsible for PKM2-dominant expression. In this study, we examined whether this machinery was conserved in the case of other PTBP1- and PKM-targeting miRNAs. We focused on the MIRs 122, 137, and 206, and investigated the expression profiles of each of these miRNAs in tissues from mouse and human organs. Also, we examined the regulatory mechanisms of PKM isoform expression by testing each of these miRNAs in human cancer cell lines. Presently, we found that brain-specific MIR137 and muscle-specific MIR206 predominantly induced PKM1 expression through direct targeting of PTBP1. Also, liver-specific MIR122 suppressed the expression of both PKM1 and PKM2, which action occurred through direct targeting of PKM to enable the expression of PKLR. Moreover, the expression levels of these miRNAs were downregulated in cancer cells that had originated from these tissues, resulting in PKM2 dominance. Our results suggest that the organ-specific distribution of miRNAs is one of the principal means by which miRNA establishes characteristics of a tissue and that dysregulation of these miRNAs results in cancer development through a change in the ratio of PKM isoform expression. Also, our results contribute to cancer diagnosis and will be useful for cancer-specific therapy for the Warburg effect in the near future.
Asunto(s)
Proteínas Portadoras/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , MicroARNs/genética , Hormonas Tiroideas/genética , Regiones no Traducidas 3' , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Especificidad de Órganos/genética , Interferencia de ARN , ARN Mensajero/genética , Hormonas Tiroideas/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
Tumor necrosis-factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-superfamily that selectively induces apoptosis through death receptors (DRs) 4 and/ or DR5 in cancer cells, without affecting normal cells. Unfortunately, many clinical studies have shown that cancer cells acquire TRAIL-resistance and thus avoid TRAIL-induced apoptosis. In the current study, we newly found that PTBP1, a splicer protein that plays an important role in energy metabolism is highly expressed in TRAIL-resistant human colon cancer DLD-1. Interestingly, silencing PTBP1 by using siRNA for PTBP1 (siR-PTBP1) resulted in a significant increase in TRAIL-sensitivity along with the switching of pyruvate kinase muscle (PKM) isoforms from PKM2 to PKM1, leading to impaired Warburg effect, because the intracellular ATP levels were significantly increased and the production of lactate decreased. Notably, siR-PTBP1 canceled the resistance by increasing the expression level of DR5 and effectively inducing the translocation of DR5 to the cell surface membrane. Also, siR-PTBP1 up-regulated the expression level of CCN1, which contributed to the enhanced sensitivity to TRAIL-induced apoptosis. These findings indicate that silencing PTBP1, thus impairing the Warburg effect positively affected TRAIL-induced apoptosis and that this splicer protein may thus serve as a possible target molecule to cancel the resistance of cancer cells to TRAIL.
Asunto(s)
Glucólisis/efectos de los fármacos , Neoplasias/patología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Acetilcisteína/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proteína 61 Rica en Cisteína/metabolismo , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Multimerización de Proteína/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Bladder cancer is one of the most difficult malignancies to control. We explored the use of a novel RNA-interference method for a driver oncogene regulating cancer specific energy metabolism by the combination treatment with a small interfering RNA (siRNA) and a microRNA. After transfection of T24 and 253JB-V cells with miR-145 and/or siR-PTBP1, we examined the effects of cell growth and gene expression by performing the trypan blue dye exclusion test, Western blot, Hoechst 33342 staining, reverse transcription polymerase chain reaction (RT-PCR), and electron microscopy. The anti-cancer effects of xenograft model mice with miR-145 and/or siR-PTBP1 were then assessed. The combination treatment induced the deeper and longer growth inhibition and reduced the levels of both mRNA and protein expression of c-Myc and polypyrimidine tract-binding protein 1 (PTBP1) more than each single treatment. Notably, the combination treatment not only impaired the cancer specific energy metabolism by inhibiting c-Myc/PTBP1/PKMs axis but also inactivated MAPK/ERK and PI3K/AKT pathways examined in vitro and in vivo. Furthermore, the combination treatment induced apoptosis or autophagy; but, in some cells, apoptotic cell death was accompanied by autophagy, because the condensation of chromatin and many autophagosomes were coexistent. This combination treatment could be a novel RNA-interference strategy through the systemic silencing of the Warburg effect-promoting driver oncogene PTBP1 in bladder cancer cells.
Asunto(s)
Apoptosis/genética , Glucólisis/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , MicroARNs/genética , Proteína de Unión al Tracto de Polipirimidina/genética , Interferencia de ARN , Neoplasias de la Vejiga Urinaria/genética , Anciano , Anciano de 80 o más Años , Animales , Western Blotting , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Modelos Genéticos , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Tratamiento con ARN de Interferencia/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/terapia , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The human DEAD/H-box RNA helicase gene DDX6 is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein has been shown to cause malignant transformation. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, ribosome assembly, and more. However, details of the regulatory mechanism of DDX6 and functions of DDX6 in cancer cells are largely unknown. On the other hand, the Warburg effect is a well-known feature of cancer cells. Pyruvate kinase in muscle (PKM), which is a rate-limiting glycolytic enzyme, has 2 isoforms, PKM1 and PKM2. It has been frequently reported that PKM2 is a tumor-specific isoform and promotes the Warburg effect. However, the functions of the PKM1 gene have been hardly mentioned. Here, we showed that DDX6 was overexpressed in colorectal cancer specimens and regulated by microRNA (miR)-124 in colon cancer cells. Also, a DDX6/c-Myc/PTB1 positive feedback circuit regulated by miR-124 was shown to be established and to contribute to maintenance of the Warburg effect. Moreover, we showed that knockdown of DDX6 induced mainly apoptosis through an imbalance of PKM gene expression, especially causing down-regulation of PKM1 in colon cancer cells. These results suggest that miR-124 is a fine tuner of the Warburg effect and that DDX6 is one of the key molecules in Warburg effect-related miR-124 targeting various genes.
RESUMEN
Resistance to chemotherapy is a crucial problem in the clinical situation. To overcome this issue, many mechanisms of chemoresistance have been elucidated so far. However, this problem still has not been solved completely. In this study, we investigated the mechanism of chemoresistance from the view of cancer metabolism-related genes, especially focusing on the expression profile of pyruvate kinase muscle (PKM) isoforms, which are rate-limiting enzymes in cancer-specific metabolism (Warburg effect). Herein, we showed that PKM1, which promotes oxidative phosphorylation (OXPHOS), was commonly up-regulated in various chemoresistant cells. To clarify the functions of PKM1 in chemoresistance, we investigated effects of PKM1 expression in DLD-1 parental, 5-FU-resistant and oxaliplatin-resistant DLD-1 cells. The overexpression of PKM1 resulted in resistance of the parental cells to 5-FU and oxaliplatin. Moreover, gene-silencing of PKM1 induced apoptosis in these cells including the resistant cells by causing a decrease in the mitochondrial membrane potential. Furthermore, combination therapy using 5-FU or oxaliplatin with siR-PKM1 was also effective against the resistant cells. Our findings should lead to the development of new agents that can cancel the chemoresistance from the view of cancer energy metabolism.
Asunto(s)
Antineoplásicos/química , Proteínas Portadoras/metabolismo , Resistencia a Antineoplásicos , Proteínas de la Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Hormonas Tiroideas/metabolismo , Apoptosis , Línea Celular Tumoral , Fluorouracilo/química , Regulación Neoplásica de la Expresión Génica , Glucólisis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Células K562 , Compuestos Organoplatinos/química , Oxaliplatino , Fosforilación Oxidativa , Fenotipo , Fase de Descanso del Ciclo Celular , Transfección , Proteínas de Unión a Hormona TiroideRESUMEN
Organic gem-dihydroperoxides (DHPs) and their derived peroxides have attracted a great deal of attention as potential anti-cancer agents. However, the precise mechanism of their inhibitory effect on tumors is unknown. To determine the mechanism of the inhibitory effects of DHPs, we examined the effects of DHPs on leukemia K562 cells. As a result, certain DHPs used in this study exhibited growth-inhibitory activity according to a clear structure-activity relationship. The most potent DHP, 12AC3O, induced apoptosis in K562 cells, but not in peripheral blood monocytes (PBMCs) or fibroblast cells. 12AC3O induced apoptosis through the intrinsic mitochondrial pathway and thereafter through the extrinsic pathway. The activity of the former pathway was partly attenuated by a JNK inhibitor. Interestingly, 12AC3O induced apoptosis by trapping a large amount of ROS, leading to an extremely lower intracellular ROS level compared with that in the cells in the steady-state condition. These results suggest that an appropriate level of intracellular ROS was necessary for the maintenance of cancer cell growth. DHPs may have a potential to be a novel anti-cancer agent with minimum adverse effects on normal cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Leucemia/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/química , Humanos , Peróxido de Hidrógeno/química , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células K562 , Leucemia/metabolismoRESUMEN
Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.
Asunto(s)
Biomarcadores de Tumor/sangre , Hemangiosarcoma/sangre , MicroARNs/sangre , Animales , Línea Celular Tumoral , Perros , Hemangiosarcoma/veterinaria , HumanosRESUMEN
Extracellular vesicles (EVs) are nanoscale entities secreted by various cells, encapsulating various nucleic acids and proteins that play important roles in cellular activities. Although rice bran is known for its richness in phytochemicals such as tocopherol and tocotrienol, the distribution of these compounds within EVs has not been extensively studied. The objective of this study was to detect and analyze the presence of vitamin E in EVs extracted from rice bran. We investigated several EV extraction methods, including rotation, vortex mixing, and ultrasonication, followed by post-extraction techniques such as ultracentrifugation, ultrafiltration, and lyophilization. Vitamin E in the EVs from rice bran was analyzed using LC-FLD. This study is the first to identify tocopherol and tocotrienol in rice bran-derived EVs. Our results indicate that ultracentrifugation followed by rotation is the most effective method for the preparation of rice bran-derived EVs. Notably, the vitamin E profile in EVs varies depending on the preparation method and differs from that in rice bran extracts. The pronounced presence of vitamin E in EVs suggests unique pharmacokinetics and underscores the potential of EVs as carriers for drug delivery systems. This study not only confirms the presence of vitamin E in EVs, but also underscores the potential of EVs and their phytochemical content for therapeutic applications.