RESUMEN
Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Humanos , Apoptosis , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Quinasas Similares a Doblecortina , Puntos de Control de la Fase G2 del Ciclo Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , AnimalesRESUMEN
Polycystic kidney disease (PKD) is a genetic disorder characterized by aberrant renal epithelial cell proliferation and formation and progressive growth of numerous fluid-filled cysts within the kidneys. Previously, we showed that there is elevated Notch signaling compared to normal renal epithelial cells and that Notch signaling contributes to the proliferation of cystic cells. Quinomycin A, a bis-intercalator peptide, has previously been shown to target the Notch signaling pathway and inhibit tumor growth in cancer. Here, we show that Quinomycin A decreased cell proliferation and cyst growth of human ADPKD cyst epithelial cells cultured within a 3D collagen gel. Treatment with Quinomycin A reduced kidney weight to body weight ratio and decreased renal cystic area and fibrosis in Pkd1RC/RC ; Pkd2+/- mice, an orthologous PKD mouse model. This was accompanied by reduced expression of Notch pathway proteins, RBPjk and HeyL and cell proliferation in kidneys of PKD mice. Quinomycin A treatments also normalized cilia length of cyst epithelial cells derived from the collecting ducts. This is the first study to demonstrate that Quinomycin A effectively inhibits PKD progression and suggests that Quinomycin A has potential therapeutic value for PKD patients.
Asunto(s)
Antibacterianos/farmacología , Quistes/tratamiento farmacológico , Modelos Animales de Enfermedad , Equinomicina/farmacología , Enfermedades Renales Poliquísticas/complicaciones , Canales Catiónicos TRPP/fisiología , Animales , Quistes/etiología , Quistes/metabolismo , Quistes/patología , Progresión de la Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in ß-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and ß-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased ß-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.
Asunto(s)
Membrana Celular/metabolismo , Citrobacter rodentium/fisiología , Infecciones por Enterobacteriaceae/genética , Epigénesis Genética , Vía de Señalización Wnt/genética , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Células HCT116 , Células HEK293 , Humanos , Ligandos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Organoides/patología , Receptores Notch/metabolismoRESUMEN
BACKGROUND & AIMS: Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice. METHODS: We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots. RESULTS: Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells. CONCLUSIONS: Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.
Asunto(s)
Antipsicóticos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Penfluridol/farmacología , Prolactina/metabolismo , Receptores de Prolactina/antagonistas & inhibidores , Animales , Antipsicóticos/uso terapéutico , Autofagia/efectos de los fármacos , Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Técnicas de Silenciamiento del Gen , Humanos , Inyecciones Intraperitoneales , Janus Quinasa 2/metabolismo , Masculino , Ratones , Páncreas/patología , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Penfluridol/uso terapéutico , Prolactina/sangre , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bladder cancer is the 9th most prevalent cancer worldwide and carries a protracted treatment course with significant patient expense, morbidity, and mortality. Over 95% of bladder cancers arise from the urothelium and invade into the underlying muscle layer before metastasizing. Trans-urethral resection and BCG therapy is the current first-line treatment for non-muscle invasive bladder cancer but carries a high rate of tumor recurrence and progression. The poor outcomes associated with advanced disease indicate the urgent need for new and improved treatment strategies. There is increasing investigation into the molecular signaling pathways involved in bladder cancer pathogenesis with the goal of uncovering potential therapeutic targets. This article reviews the major signaling pathways implicated in bladder cancer, including PI3K/AKT/mTOR, Ras/Raf/MEK/MAPK, NF-κB, Wnt/ß-catenin, Notch, Hedgehog, Hippo, JAK/STAT, and TGF-ß as well as major cellular receptors central to cancer pathophysiology, including EGFR, Her2, FGFR, and VEGF. We also discuss various naturally occurring phytochemicals that show evidence of targeting these molecular pathways including curcumin, resveratrol, green tea polyphenols, sulforaphane, erucin, genistein, genipin, baicalein, quercetin, isoquercitin, vitamin E, parthenolide, dioscin, triptolide, kaempferol, pterostilbene, isoliquiritigenin, and escin. This review highlights the potential use of these compounds in treatment of bladder cancer.
Asunto(s)
Neoplasias de la Vejiga Urinaria , Genisteína , Humanos , Fosfatidilinositol 3-Quinasas , Fitoquímicos/farmacología , Fitoquímicos/uso terapéutico , Transducción de Señal , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
BACKGROUND & AIMS: The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. METHODS: We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. RESULTS: Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. CONCLUSIONS: We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.
Asunto(s)
Carcinogénesis/genética , Carcinoma Ductal Pancreático/genética , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Metilación de ADN , Conjuntos de Datos como Asunto , Quinasas Similares a Doblecortina , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Ratones , Persona de Mediana Edad , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Supervivencia , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously reported that ionizing radiation (IR) mediates cell death through the induction of CUGBP elav-like family member 2 (CELF2), a tumor suppressor. CELF2 is an RNA binding protein that modulates mRNA stability and translation. Since IR induces autophagy, we hypothesized that CELF2 regulates autophagy-mediated colorectal cancer (CRC) cell death. For clinical relevance, we determined CELF2 levels in The Cancer Genome Atlas (TCGA). Role of CELF2 in radiation response was carried out in CRC cell lines by immunoblotting, immunofluorescence, autophagic vacuole analyses, RNA stability assay, quantitative polymerase chain reaction and electron microscopy. In vivo studies were performed in a xenograft tumor model. TCGA analyses demonstrated that compared to normal tissue, CELF2 is expressed at significantly lower levels in CRC, and is associated with better overall 5-year survival in patients receiving radiation. Mechanistically, CELF2 increased levels of critical components of the autophagy cascade including Beclin-1, ATG5, and ATG12 by modulating mRNA stability. CELF2 also increased autophagic flux in CRC. IR significantly induced autophagy in CRC which correlates with increased levels of CELF2 and autophagy associated proteins. Silencing CELF2 with siRNA, mitigated IR induced autophagy. Moreover, knockdown of CELF2 in vivo conferred tumor resistance to IR. These studies elucidate an unrecognized role for CELF2 in inducing autophagy and potentiating the effects of radiotherapy in CRC.
Asunto(s)
Autofagia/fisiología , Proteínas CELF/metabolismo , Supervivencia Celular/efectos de la radiación , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/radioterapia , Proteínas del Tejido Nervioso/metabolismo , Animales , Proteína 12 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Proteínas CELF/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Células HCT116 , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Proteínas del Tejido Nervioso/genética , Pronóstico , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Radiación Ionizante , Trasplante HeterólogoRESUMEN
Breast cancer is the most common form of cancer diagnosed in women worldwide and the second leading cause of cancer-related deaths in the USA. Despite the development of newer diagnostic methods, selective as well as targeted chemotherapies and their combinations, surgery, hormonal therapy, radiotherapy, breast cancer recurrence, metastasis and drug resistance are still the major problems for breast cancer. Emerging evidence suggest the existence of cancer stem cells (CSCs), a population of cells with the capacity to self-renew, differentiate and be capable of initiating and sustaining tumor growth. In addition, CSCs are believed to be responsible for cancer recurrence, anticancer drug resistance, and metastasis. Hence, compounds targeting breast CSCs may be better therapeutic agents for treating breast cancer and control recurrence and metastasis. Naturally occurring compounds, mainly phytochemicals have gained immense attention in recent times because of their wide safety profile, ability to target heterogeneous populations of cancer cells as well as CSCs, and their key signaling pathways. Therefore, in the present review article, we summarize our current understanding of breast CSCs and their signaling pathways, and the phytochemicals that affect these cells including curcumin, resveratrol, tea polyphenols (epigallocatechin-3-gallate, epigallocatechin), sulforaphane, genistein, indole-3-carbinol, 3, 3'-di-indolylmethane, vitamin E, retinoic acid, quercetin, parthenolide, triptolide, 6-shogaol, pterostilbene, isoliquiritigenin, celastrol, and koenimbin. These phytochemicals may serve as novel therapeutic agents for breast cancer treatment and future leads for drug development.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Supervivencia Celular , Humanos , Células Madre Neoplásicas/fisiología , Fitoquímicos/farmacología , Fitoquímicos/uso terapéutico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéuticoRESUMEN
Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea(AR; Hollyhock) is an ornamental plant with known anti-inflammatory properties. This study explored its role as an anticancer agent. The AR seed extract (AR extract) inhibited proliferation and colony formation in a dose- and time-dependent manner and promoted apoptosis as was evidenced by cleavage of PARP and increased expression of Bax accompanying reduced levels of BCL-xl protein in HCT116 and SW480 cells, respectively. In addition, AR extract-arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. AR extract-treated cells exhibited reduced number and size of colonospheres in a dose-dependent manner concomitant with decreases in cancer stem cell (CSC) markers ALDH1A1 and Dclk1. Relative levels of ß-catenin, Notch-ICD, Hes1 and EZH2 were also attenuated by AR extract. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown.In vivo, AR extract attenuated tumor growth due probably to reduced levels of EZH2, ß-catenin, CyclinD1 and Ki-67 along with reduced levels of CSC markers. Since partial purification via HPLC yielded a prominent peak, efforts are underway to identify the active ingredient(s). Taken together, the results clearly suggest that AR extract/active component(s) can be an effective preventative/therapeutic agent to target colon cancer.
Asunto(s)
Neoplasias del Colon/patología , Epigénesis Genética , Células Madre Neoplásicas/patología , Plantas , Transducción de Señal , HumanosRESUMEN
Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. It arises from loss of intestinal epithelial homeostasis and hyperproliferation of the crypt epithelium. In order to further understand the pathogenesis of CRC it is important to further understand the factors regulating intestinal epithelial proliferation and more specifically, regulation of the intestinal epithelial stem cell compartment. Here, we investigated the role of the RNA binding protein RBM3 in stem cell homeostasis in colorectal cancers. Using a doxycycline (Dox) inducible RBM3 overexpressing cell lines HCT 116 and DLD-1, we measured changes in side population (SP) cells that have high xenobiotic efflux capacity and increased capacity for self-renewal. In both cell lines, RBM3 induction showed significant increases in the percentage of side population cells. Additionally, we observed increases in spheroid formation and in cells expressing DCLK1, LGR5 and CD44Hi . As the Wnt/ß-catenin signaling pathway is important for both physiologic and cancer stem cells, we next investigated the effects of RBM3 overexpression on ß-catenin activity. RBM3 overexpression increased levels of nuclear ß-catenin as well as TCF/LEF transcriptional activity. In addition, there was inactivation of GSK3ß leading to decreased ß-catenin phosphorylation. Pharmacologic inhibition of GSK3ß using (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO) also recapitulates the RBM3 induced ß-catenin activity. In conclusion, we see that RNA binding protein RBM3 induces stemness in colorectal cancer cells through a mechanism involving suppression of GSK3ß activity thereby enhancing ß-catenin signaling. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Neoplasias Colorrectales/patología , Células Madre Neoplásicas/citología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Doxiciclina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HCT116 , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fosforilación , Proteínas Wnt/metabolismo , Vía de Señalización WntRESUMEN
Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self-renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor-α-Converting Enzyme (TACE) and γ-secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch-2. In addition, there was a reduction in the expression of downstream target proteins, Hes-1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ-secretase complex proteins in melanoma cells. To confirm that suppression of Notch-2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes-1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling.
Asunto(s)
Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Receptor Notch2/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Autofagia/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclina D1/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción HES-1RESUMEN
Prolactin (PRL) is a secretory cytokine produced by various tissues. Binding to the cognate PRL receptor (PRLR), it activates intracellular signaling via janus kinase (JAK), extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) proteins. PRL regulates diverse activities under normal and abnormal conditions, including malignancies. Previous clinical data suggest serum PRL levels are elevated in colorectal cancer (CRC) patients. In this study, we first determined the expression of PRL and PRLR in colon cancer tissue and cell lines. Higher levels of PRLR expression were observed in the cancer cells and cell lines compared with normal colonic epithelial cells. Incubation of colon cancer cells with PRL-induced JAK2, STAT3 and ERK1/2 phosphorylation and increased expression of Jagged 1, which is a Notch-1 receptor ligand. Notch signaling regulates CRC stem cell population. We observed increased accumulation of the cleaved/active form of Notch-1 receptor (Notch intracellular domain) and increased expression of Notch responsive genes HEY1, HES1 and stem cell marker genes DCLK1, LGR5, ALDH1 and CD44. Finally, inhibiting PRL induced JAK2-STAT3 and JAK2-ERK1/2 using AG490 and PD98059, respectively, leads to complete abrogation of Notch signaling, suggesting a role for this pathway in regulating CRC stem cells. Together, our results demonstrate that cytokine signaling induced by PRL is active in colorectal cancers and may provide a novel target for therapeutic intervention.
Asunto(s)
Neoplasias del Colon/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Janus Quinasa 2/metabolismo , Células Madre Neoplásicas/metabolismo , Prolactina/metabolismo , Receptores Notch/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Secuencia de Bases , Neoplasias del Colon/metabolismo , Cartilla de ADN , Humanos , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
Musashi1 (Msi1) is a highly conserved RNA-binding protein that is required during the development of the nervous system. Msi1 has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation, and has also been implicated in tumorigenesis, being highly expressed in multiple tumor types. We analyzed Msi1 expression in a large cohort of medulloblastoma samples and found that Msi1 is highly expressed in tumor tissue compared with normal cerebellum. Notably, high Msi1 expression levels proved to be a sign of poor prognosis. Msi1 expression was determined to be particularly high in molecular subgroups 3 and 4 of medulloblastoma. We determined that Msi1 is required for tumorigenesis because inhibition of Msi1 expression by small-interfering RNAs reduced the growth of Daoy medulloblastoma cells in xenografts. To characterize the participation of Msi1 in medulloblastoma, we conducted different high-throughput analyses. Ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-chip) was used to identify mRNA species preferentially associated with Msi1 protein in Daoy cells. We also used cluster analysis to identify genes with similar or opposite expression patterns to Msi1 in our medulloblastoma cohort. A network study identified RAC1, CTGF, SDCBP, SRC, PRL, and SHC1 as major nodes of an Msi1-associated network. Our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.
Asunto(s)
Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Redes Reguladoras de Genes/genética , Genes Relacionados con las Neoplasias/genética , Meduloblastoma/genética , Meduloblastoma/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genoma Humano/genética , Células HEK293 , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Inducción de Remisión , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116 is known to have a mutation in the hMLH1 mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer. This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3). We first determined that the combination of honokiol and γ-irradiation treatment resulted in dose-dependent inhibition of proliferation and colony formation in both cell lines. However, the effects were more pronounced in HCT116 cells. Similarly, the combination induced higher levels of apoptosis (caspase 3 activation, Bax to Bcl2 ratio) in the HCT116 cells compared with HCT116-CH3 cells. Cell cycle analyses revealed higher levels of dead cells in HCT116 cells. The combination treatment reduced expression of cyclin A1 and D1 and increased phosphorylated p53 in both cell lines, although there were significantly lower amounts of phosphorylated p53 in the HCT116-CH3 cells, suggesting that high levels of hMLH1 reduce radiosensitivity. These data demonstrate that honokiol is highly effective in radiosensitizing colorectal cancer cells, especially those with a mismatch repair defect.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Compuestos de Bifenilo/farmacología , Proliferación Celular/efectos de los fármacos , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Lignanos/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Células HCT116 , Humanos , Tolerancia a Radiación/genética , Células Tumorales CultivadasRESUMEN
Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of -7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Compuestos de Bifenilo/farmacología , Carcinogénesis/patología , Neoplasias del Colon/patología , Lignanos/farmacología , Células Madre Neoplásicas/patología , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colitis/complicaciones , Quinasas Similares a Doblecortina , Regulación hacia Abajo/efectos de los fármacos , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones Endogámicos ICR , Modelos Biológicos , Células Madre Neoplásicas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Proteínas Señalizadoras YAPRESUMEN
Spiropyrans have been extensively investigated because of their thermo- and photochromic characteristics, but their biotherapeutic properties have not been explored much. We report anti-proliferative properties of a novel 3,3'-azadimethylene dinaphthospiropyran 11. Dibenzospiropyrans and dinaphthospiropyrans were synthesized by a simple and expedient method using acid-catalyzed aldol condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde, respectively, with cyclic ketones. Together with structural elucidation by 2D NMR and X-ray crystallography studies, we provide a putative mechanism for their formation. Compound 11 showed solvatochromism and exhibited altered spectral characteristics depending on the pH. In acidic conditions, 11 remains in open form, whereas upon alkalinization it reverts back to closed form. Based on the in vitro anti-proliferative activity in H441, HCT-116, MiaPaCa-2, and Panc-1 cancer cell lines, 11 was submitted to further investigation. It reduced HCT116 colonosphere formation and demonstrated induction of caspase cascade, suggesting apoptosis. In vitro proliferation assays also suggested that HCl and trifluoroacetate salts of 11 are more effective. Treatment of mice carrying HCT-116 xenografts with 11 (5 µg/day, intraperitoneal for 3 weeks) suppressed tumor growth by 62%. Overall, the results reveal a new series of structurally complex, but relatively easy to synthesize molecules of which compound 11 represents a lead for anticancer development.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzopiranos/química , Neoplasias del Colon/tratamiento farmacológico , Indoles/química , Nitrocompuestos/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Benzopiranos/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Ratones , Conformación Molecular , Nitrocompuestos/farmacología , Nitrocompuestos/uso terapéutico , Trasplante HeterólogoRESUMEN
Despite the recent launch of tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) is contained in a number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed, including chelation of iron and inhibition of iron-dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess the in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney-to-body weight ratios of PKD mice. The CPX-O treatment was also associated with decreased cell proliferation, decreased cystic area, and improved renal function. Ferritin levels were markedly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker nuclear receptor coactivator 4, which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy, which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.
Asunto(s)
Antifúngicos/farmacología , Ciclopirox/farmacología , Quistes , Ferritinas/metabolismo , Riñón/efectos de los fármacos , Enfermedades Renales Poliquísticas , Animales , Antifúngicos/uso terapéutico , Proliferación Celular , Ciclopirox/uso terapéutico , Colágeno , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Ratones Endogámicos C57BL , Coactivadores de Receptor Nuclear/metabolismo , Tamaño de los Órganos , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Riñón Poliquístico Autosómico DominanteRESUMEN
A new and readily available pentafluorothiophenyl-substituted N-methyl-piperidone curcuminoid 1a was prepared and investigated for its anti-proliferative, pro-apoptotic and cancer stem cell-differentiating activities against a panel of human tumor cell lines derived from various tumor entities. The compound 1a was highly anti-proliferative and reached IC50 values in the nanomolar concentration range. 1a was superior to the known anti-tumorally active curcuminoid EF24 (2) and its known N-ethyl-piperidone analog 1b in all tested tumor cell lines. Furthermore, 1a induced a noticeable increase of intracellular reactive oxygen species in HT-29 colon adenocarcinoma cells, which possibly leads to a distinct increase in sub-G1 cells, as assessed by cell cycle analysis. A considerable activation of the executioner-caspases 3 and 7 as well as nuclei fragmentation, cell rounding, and membrane protrusions suggest the triggering of an apoptotic mechanism. Yet another effect was the re-organization of the actin cytoskeleton shown by the formation of stress fibers and actin aggregation. 1a also caused cell death in the adherently cultured glioblastoma cell lines U251 and Mz54. We furthermore observed that 1a strongly suppressed the stem cell properties of glioma stem-like cell lines including one primary line, highlighting the potential therapeutic relevance of this new compound.
Asunto(s)
Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/farmacología , Diarilheptanoides/síntesis química , Diarilheptanoides/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Curcumina/síntesis química , Curcumina/farmacología , Relación Dosis-Respuesta a Droga , Células HCT116 , Células HT29 , Humanos , Células MCF-7 , Pez CebraRESUMEN
BACKGROUND & AIMS: MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression. The tumor suppressor miRNA let-7a has been reported to be inhibited posttranscriptionally in embryonic stem cells and in human cancers. Microtubule-associated kinase DCAMKL-1 is a putative intestinal stem cell marker that is expressed in Apc(Min/+) adenomas. We investigated the role of DCAMKL-1 on expression of let-7a miRNA and the oncogene c-Myc and in tumorigenesis. METHODS: Human tissue microassay slides were immunostained for DCAMKL-1. HCT116 and SW480 cells were transfected with DCAMKL-1 small interfering RNA (siRNA) (si-DCAMKL-1) and analyzed for DCAMKL-1, c-Myc (using immunoblot and real-time reverse-transcription polymerase chain reaction [RT-PCR]), and pri-let-7a miRNA (using real-time RT-PCR) levels. A liposomal preparation of si-DCAMKL-1 was administered into HCT116 xenografts in nude mice, and tumor volumes were measured. A luciferase reporter assay, with a plasmid containing a let-7a-binding site at the 3' untranslated region, was utilized to measure let-7a in cell lines. Cells were isolated from normal mouse intestine using DCAMKL-1 and fluorescence-activated cell sorting (FACS) and subjected to pri-let-7a miRNA analysis. RESULTS: Expression of DCAMKL-1 was increased in human colorectal cancers. siRNA-mediated blockade of DCAMKL-1 resulted in H tumor xenograft growth arrest, increased pri-let-7a miRNA, a corresponding decrease in luciferase activity, and decreased expression of the oncogene c-Myc. DCAMKL-1(+) cells isolated by FACS demonstrated a significant decrease in pri-let-7a miRNA, compared with more differentiated cells. CONCLUSIONS: DCAMKL-1 is a negative regulator of let-7a miRNA biogenesis in intestinal stem and colorectal cancer cells; it could represent a novel target for anti-cancer stem cell-based strategies.
Asunto(s)
Neoplasias Colorrectales/genética , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , MicroARNs/genética , Análisis de Varianza , Animales , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/fisiopatología , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Inmunohistoquímica , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Ratones , Ratones Desnudos , MicroARNs/biosíntesis , MicroARNs/metabolismo , Probabilidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Trasplante Heterólogo , Carga Tumoral/genéticaRESUMEN
Trivalent arsenic [As(III)] is currently approved by the FDA for the treatment of chronic and acute leukemias. However, As(III) has also demonstrated damaging effects on human health, including development of cardiovascular disease, diabetes, and cancer. Further, As(III) is a potent angiogenic agent. In this context, curcumin, an active ingredient in the dietary agent turmeric, has demonstrated potent antiproliferative, antiinflammatory, and antiangiogenic properties. In this report, we have shown that both curcumin and turmeric inhibit expression of vascular endothelial growth factor in HCT-116 human colon cancer cells exposed to As(III). Further, in the chicken chorioallantoic membrane assay model, treatment with low As(III) concentrations results in extensive increase in blood vessel density, which, however, is reduced in the presence of curcumin or turmeric. Collectively, the findings reported here strongly suggest that turmeric and curcumin can dramatically attenuate the process of angiogenesis induced by low As(III) concentrations.