RESUMEN
Prostate cancer is the second most frequent cancer and the fifth leading cause of cancer death among men worldwide. While the five-year survival in local and regional prostate cancer is higher than 99%, it falls to about 28% in advanced metastatic prostate cancer. The ether lipid edelfosine is considered the prototype of a family of promising antitumor drugs collectively named as alkylphospholipid analogs. Here, we found that edelfosine was the most potent alkylphospholipid analog in inducing apoptosis in three different human prostate cancer cell lines (LNCaP, PC3, and DU145) with distinct androgen dependency, and differing in tumor suppressor phosphatase and tensin homolog (PTEN) and p53 status. Edelfosine accumulated in the endoplasmic reticulum of prostate cancer cells, leading to endoplasmic reticulum stress and cell death in the three prostate cancer cells. Inhibition of autophagy potentiated the pro-apoptotic activity of edelfosine in LNCaP and PC3 cells, where autophagy was induced as a survival response. Edelfosine induced a slight and transient inhibition of AKT in PTEN-negative LNCaP and PC3 cells, but not in PTEN-positive DU145 cells. Daily oral administration of edelfosine in murine prostate restricted AKT kinase transgenic mice, expressing active AKT in a prostate-specific manner, and in a DU145 xenograft mouse model resulted in significant tumor regression and apoptosis in tumor cells. Taken together, these results show a significant in vitro and in vivo antitumor activity of edelfosine against prostate cancer, and highlight the endoplasmic reticulum as a novel and promising therapeutic target in prostate cancer.
RESUMEN
The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Berberina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Melanoma/fisiopatología , Mitocondrias/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/fisiología , Transducción de Señal/efectos de los fármacos , Nucleótidos de Adenina/metabolismo , Western Blotting , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Citocromos c/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Citometría de Flujo , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Humanos , Microscopía Confocal , Proteínas Proto-Oncogénicas B-raf/biosíntesis , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.
Asunto(s)
Bronquiolos/patología , Neoplasias Pulmonares/patología , Metaloproteinasa 7 de la Matriz/fisiología , Lesiones Precancerosas/patología , Alveolos Pulmonares/patología , Animales , Bronquiolos/enzimología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Humanos , Neoplasias Pulmonares/enzimología , Metaloproteinasa 7 de la Matriz/genética , Ratones , Ratones Transgénicos , Lesiones Precancerosas/enzimología , Alveolos Pulmonares/enzimologíaRESUMEN
MicroRNAs (miRNAs, miRs) are short non-coding post-transcriptional regulators of gene expression in normal physiology and disease. Acute myeloid leukemia is characterized by accumulation of malignantly transformed immature myeloid precursors, and differentiation therapy, used to overcome this differentiation blockage, has become a successful therapeutic option. The human HL-60 acute leukemia cell line serves as a cell culture model for granulocytic maturation, and dimethyl sulfoxide (DMSO) incubation leads to its differentiation towards neutrophil-like cells, as assessed by biochemical, functional and morphological parameters. DMSO-induced HL-60 cell differentiation constitutes an excellent model to examine molecular processes that turn a proliferating immortal leukemic cell line into mature non-proliferating and apoptosis-prone neutrophil-like end cells. By performing genome-wide miRNA profiling and functional assays, we have identified a signature of 86 differentially expressed canonical miRNAs (51 upregulated; 35 downregulated) during DMSO-induced granulocytic differentiation of HL-60 cells. Quantitative real-time PCR was used to validate miRNA expression. Among these differentially expressed canonical miRNAs, we found miR-125a-5p upregulation and miR-17-92 cluster downregulation acted as major regulators of granulocytic differentiation in HL-60 cells. Enforced expression of miR-125a-5p promoted granulocytic differentiation in HL-60 cells, whereas miR-17-92 ectopic expression inhibited DMSO-induced HL-60 granulocytic differentiation. Ectopic expression of miR-125a-5p also promoted granulocytic differentiation in human acute promyelocytic leukemia NB4 cells, as well as in naïve human primary CD34+-hematopoietic progenitor/stem cells. These findings provide novel molecular insights into the identification of miRNAs regulating granulocytic differentiation of human leukemia cells and normal CD34+-hematopoietic progenitor/stem cells, and may assist in the development of novel miRNA-targeted therapies for leukemia.
RESUMEN
Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells in vitro. In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells. Furthermore, we found that Pimozide also promoted apoptosis as demonstrated by cell cycle arrest and induction of double-strand DNA breaks but did not result in any effect in the non-transformed MCF10A breast cell line. In order to shed new lights into the molecular pathways affected by Pimozide, we show that Pimozide downregulated RAN GTPase and AKT at both protein and mRNA levels and inhibited the AKT signaling pathway in MDA-MB-231 breast cancer cells. Pimozide also inhibited the epithelial mesenchymal transition and cell migration and downregulated the expression of MMPs. Administration of Pimozide showed a potent in vivo antitumor activity in MDA-MB-231 xenograft animal model and reduced the number of lung metastases by blocking vascular endothelial growth factor receptor 2. Furthermore, Pimozide inhibited myofibroblast formation as evaluated by the reduction in α-smooth muscle actin containing cells. Thus, Pimozide might inhibit tumor development by suppressing angiogenesis and by paracrine stimulation provided by host reactive stromal cells. These results demonstrate a novel in vitro and in vivo antitumor activity of Pimozide against breast and lung cancer cells and provide the proof of concept for a putative Pimozide as a novel approach for cancer therapy.
RESUMEN
Ran is a small ras-related GTPase and is highly expressed in aggressive breast carcinoma. Overexpression induces malignant transformation and drives metastatic growth. We have designed a novel series of anti-Ran-GTPase peptides, which prevents Ran hydrolysis and activation, and although they display effectiveness in silico, peptide activity is suboptimal in vitro due to reduced bioavailability and poor delivery. To overcome this drawback, we delivered an anti-Ran-GTPase peptide using encapsulation in PLGA-based nanoparticles (NP). Formulation variables within a double emulsion solvent evaporation technique were controlled to optimise physicochemical properties. NP were spherical and negatively charged with a mean diameter of 182-277nm. Peptide integrity and stability were maintained after encapsulation and release kinetics followed a sustained profile. We were interested in the relationship between cellular uptake and poly(ethylene glycol) (PEG) in the NP matrix, with results showing enhanced in vitro uptake with increasing PEG content. Peptide-loaded, pegylated (10% PEG)-PLGA NP induced significant cytotoxic and apoptotic effects in MDA-MB-231 breast cancer cells, with no evidence of similar effects in cells pulsed with free peptide. Western blot analysis showed that encapsulated peptide interfered with the proposed signal transduction pathway of the Ran gene. Our novel blockade peptide prevented Ran activation by blockage of regulator of chromosome condensation 1 (RCC1) following peptide release directly in the cytoplasm once endocytosis of the peptide-loaded nanoparticle has occurred. RCC1 blockage was effective only when a nanoparticulate delivery approach was adopted.
Asunto(s)
Neoplasias de la Mama , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/química , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Nanopartículas/química , Proteínas Nucleares/antagonistas & inhibidores , Poliésteres/química , Polietilenglicoles/química , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Femenino , Proteínas Activadoras de GTPasa/administración & dosificación , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Nanopartículas/administración & dosificación , Proteínas Nucleares/fisiología , Poliésteres/administración & dosificación , Polietilenglicoles/administración & dosificaciónRESUMEN
Tumour suppressor proteins, such as p53, BRCA1, and ABC, play key roles in preventing the development of a malignant phenotype, but those that function as transcriptional regulators need to enter the nucleus in order to function. The export of proteins between the nucleus and cytoplasm is complex. It occurs through nuclear pores and exported proteins need a nuclear export signal (NES) to bind to nuclear exportin proteins, including CRM1 (Chromosomal Region Maintenance protein 1), and the energy for this process is provided by the RanGTP/RanGDP gradient. Due to the loss of DNA repair and cell cycle checkpoints, drug resistance is a major problem in cancer treatment, and often an initially successful treatment will fail due to the development of resistance. An important mechanism underlying resistance is nuclear export, and a number of strategies that can prevent nuclear export may reverse resistance. Examples include inhibitors of CRM1, antibodies to the nuclear export signal, and alteration of nuclear pore structure. Each of these are considered in this review.
RESUMEN
It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.
Asunto(s)
Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Proteína de Unión al GTP ran/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Neoplasias/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP ran/genéticaRESUMEN
Ewing's sarcoma (ES) is the second most common bone cancer in children and young people. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the prototype of a family of synthetic antitumor compounds, collectively known as alkylphospholipid analogs (APLs). We have found that APLs ranked edelfosine>perifosine>erucylphosphocholine>miltefosine for their capacity to promote apoptosis in ES cells. Edelfosine accumulated in the endoplasmic reticulum (ER) and triggered an ER stress response that eventually led to caspase-dependent apoptosis in ES cells. This apoptotic response involved mitochondrial-mediated processes, with cytochrome c release, caspase-9 activation and generation of reactive oxygen species. Edelfosine-induced apoptosis was also dependent on sustained c-Jun NH2-terminal kinase activation. Oral administration of edelfosine showed a potent in vivo antitumor activity in an ES xenograft animal model. Histochemical staining gave evidence for ER stress response and apoptosis in the ES tumors isolated from edelfosine-treated mice. Edelfosine showed a preferential action on ES tumor cells as compared to non-transformed osteoblasts, and appeared to be well suited for combination therapy regimens. These results demonstrate in vitro and in vivo antitumor activity of edelfosine against ES cells that is mediated by caspase activation and ER stress, and provide the proof of concept for a putative edelfosine- and ER stress-mediated approach forES treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Retículo Endoplásmico/metabolismo , Éteres Fosfolípidos/uso terapéutico , Sarcoma de Ewing/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis , Humanos , Ratones , Ratones SCID , Éteres Fosfolípidos/administración & dosificación , Éteres Fosfolípidos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer is one of the most prevalent malignancies worldwide. It consists of a group of tumor cells that have the ability to grow uncontrollably, overcome replicative senescence (tumor progression) and metastasize within the body. Metastases are processes that consist of an array of complex gene dysregulation events. Although these processes are still not fully understood, the dysregulation of a number of key proteins must take place if the tumor cells are to disseminate and metastasize. It is now widely accepted that future effective and innovative treatments of cancer metastasis will have to encompass all the major components of malignant transformation. For this reason, much research is now being carried out into the mechanisms that govern the malignant transformation processes. Recent research has identified key genes involved in the development of metastases, as well as their mechanisms of action. A detailed understanding of the encoded proteins and their interrelationship generates the possibility of developing novel therapeutic approaches. This review will focus on a select group of proteins, often deregulated in breast cancer metastasis, which have shown therapeutic promise, notably, EMT, E-cadherin, Osteopontin, PEA3, Transforming Growth Factor Beta (TGF-ß) and Ran.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Neoplasias/metabolismo , Animales , Femenino , Humanos , Metástasis de la NeoplasiaRESUMEN
BACKGROUND: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites. METHODOLOGY/PRINCIPAL FINDINGS: We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis. CONCLUSIONS/SIGNIFICANCE: Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.
Asunto(s)
Antineoplásicos/farmacología , Antiparasitarios/farmacología , Éteres Fosfolípidos/farmacología , Esquistosomiasis mansoni/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Antiparasitarios/uso terapéutico , Apoptosis/efectos de los fármacos , Femenino , Ratones , Éteres Fosfolípidos/uso terapéutico , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Schistosoma mansoni/efectos de los fármacosRESUMEN
Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFP-expressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high- and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies.
Asunto(s)
Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones EndogámicosRESUMEN
The basic helix-loop-helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas.