RESUMEN
Unlike other malignancies, ovarian cancer (OC) creates a complex tumor microenvironment with distinctive peritoneal ascites consisting of a mixture of several immunosuppressive cells which impair the ability of the patient's immune system to fight the disease. The poor survival rates observed in advanced stage OC patients and the lack of effective conventional therapeutic options have been attributed in large part to the immature dendritic cells (DCs), IL-10 secreting regulatory T cells, tumor-associated macrophages, myeloid-derived suppressor cells, and cancer stem cells that secrete inhibitory cytokines. This review highlights the critical role played by the intraperitoneal presence of IL-10 in the generation of an immunosuppressive tumor microenvironment. Further, the effect of antibody neutralization of IL-10 on the efficacy of DC and chimeric antigen receptor T-cell vaccines will be discussed. Moreover, we will review the influence of IL-10 in the promotion of cancer stemness in concert with the NF-κB signaling pathway with regard to OC progression. Finally, understanding the role of IL-10 and its crosstalk with various cells in the ascitic fluid may contribute to the development of novel immunotherapeutic approaches with the potential to kill drug-resistant OC cells while minimizing toxic side effects.
Asunto(s)
Interleucina-10 , Neoplasias Ováricas , Carcinoma Epitelial de Ovario , Células Dendríticas , Femenino , Humanos , Neoplasias Ováricas/terapia , Transducción de Señal , Microambiente TumoralRESUMEN
BACKGROUND: We have previously demonstrated in vitro cytotoxicity of mesothelin-chimeric antigen receptor autologous T cells against pancreatic cancer cells using lentiviral vectors, but these vectors pose safety concerns. Here, we incorporated Sleeping Beauty and minicircle design enhancements into interleukin-2-secreting natural NK-92MI cells to eliminate both bacterial and viral components and address inhibition by the tumor microenvironment. METHODS: Parental (conventional deoxyribonucleic acid)-mesothelin-chimeric antigen receptor and minicircle-mesothelin-chimeric antigen receptor vectors were electroporated into NK-92MI cells and engraftment was visualized by immunofluorescence analysis with protein-L staining. Interferon-γ and granzyme B secretion were measured by enzyme-linked immunosorbent assay from cocultures of parental-mesothelin-chimeric antigen receptors and minicircle-mesothelin-chimeric antigen receptors with human pancreatic cancer cells, and cytotoxicity of chimeric antigen receptor NK-92MI cells was tested against three pancreatic cancer cell lines. RESULTS: Cloning of mesothelin-chimeric antigen receptor Sleeping Beauty into a minicircle vector removed its bacterial backbone and reduced its size with improved electroporation efficiency. Chimeric antigen receptor engraftment, Interferon-γ and granzyme B secretion, and specific lysis against all three pancreatic cancer lines were significantly increased with minicircle-mesothelin-chimeric antigen receptor versus parental-mesothelin-chimeric antigen receptor NK-92MI cells. CONCLUSION: We provide proof of concept that allogeneic mesothelin-chimeric antigen receptor NK-92MI cells with hybrid Sleeping Beauty and minicircle technologies provide increased engraftment and cytotoxicity in vitro with potential safety benefits when translated to the clinical arena.
Asunto(s)
Muerte Celular/inmunología , Proteínas Ligadas a GPI/farmacología , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/inmunología , Neoplasias Pancreáticas/patología , Receptores Quiméricos de Antígenos/inmunología , Línea Celular Tumoral , Electroporación/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Células Asesinas Naturales/efectos de los fármacos , Mesotelina , Neoplasias Pancreáticas/terapia , Sensibilidad y Especificidad , Microambiente TumoralRESUMEN
BACKGROUND: Pancreatic cancer cells are known to shield themselves from immunosurveillance by secreting immune inhibitory cytokines such as Interleukin-10. Using mesothelin, a differentiating antigen that is overexpressed in pancreatic cancer, we assessed the negative effect of the tumor microenvironment on chimeric antigen receptor T cell-based immunotherapy and its reversal via depletion of Interleukin-10. METHODS: T cells cultured in pancreatic cancer-cell-conditioned medium were transduced with lentiviruses encoding mesothelin-chimeric antigen receptor in the presence or absence of anti-Interleukin-10-blocking antibody. RESULTS: Coculture supernatants of conditioned medium displayed significant inhibition of interferon γ and granzyme B secretion, both of which are crucial for induction of target cell cytotoxicity. In contrast, this inhibition was restored toward baseline when conditioned medium was Interleukin-10- depleted (p < .05 for both interferon γ and granzyme B). In addition, we observed a significant decrease in mesothelin-chimeric antigen receptor T cell-induced cytotoxicity of BxPC-3 target cells in the presence of conditioned medium. Furthermore, we observed a partial blunting of this inhibition when Interleukin-10 was depleted from the conditioned medium. CONCLUSION: Substantial reversal of tumor-derived immunosuppression may be achieved by blocking Interleukin-10 in the local microenvironment, allowing for more effective cytotoxicity of mesothelin-engrafted chimeric antigen receptor T cells and enhancing the potential for clinical application.
Asunto(s)
Proteínas Ligadas a GPI/farmacología , Interleucina-10/fisiología , Neoplasias Pancreáticas/patología , Linfocitos T/fisiología , Microambiente Tumoral/fisiología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Humanos , Inmunoterapia , Mesotelina , Receptores de Antígenos de Linfocitos TAsunto(s)
Antígenos de Neoplasias/inmunología , Péptidos de Penetración Celular/inmunología , Inmunoterapia/métodos , Proteínas de Neoplasias/inmunología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Linfocitos T Citotóxicos/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Citocinas/metabolismo , HumanosRESUMEN
PURPOSE: MicroRNA (miR)-26a has been identified as a tumor suppressor in pancreatic cancer cells. Although wild-type p53 controls cell-cycle progression, its mutant form normally present in pancreatic cancer loses this capability. Phosphorylation is known to restore wild-type activity to mutant p53. We, therefore, examined whether miR-26a treatment can restore wild-type functions of mutant p53 via phosphorylation, resulting in inhibition of cell growth. METHODS: The human pancreatic cancer cell line BxPc-3 harboring mutant p53 was used for colony formation, cell-cycle, and Western blotting assays. Gene profile analysis was conducted after transfection with pre-miR-26a. RESULTS: miR-26a expression significantly decreased cell proliferation by 80% along with marked inhibition of colony formation and cell migration. Cell-cycle inhibition at the G0/G1 interface was observed along with enhanced drug retention and increased chemosensitivity to gemcitabine. Mutant p53 was phosphorylated rapidly at its Ser9 and Ser392 residues, but not at Ser15 or Ser20. Gene profile analysis of pre-miR-26a-transfected cells showed a significant increase in gene transcripts promoting apoptosis and p53 activation, with decreased levels of genes involved in cell-cycle progression. CONCLUSION: Delivery of miR-26a may represent a novel strategy for inhibiting pancreatic cancer growth, at least in part by enhancing phosphorylation of mutant p53 to restore its wild-type functions.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proliferación Celular/fisiología , MicroARNs/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Ciclo Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Células Madre Neoplásicas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosforilación , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
IMPORTANCE: In conjunction with chemotherapy, immunotherapy with dendritic cells (DCs) may eliminate minimal disease burden by generating cytotoxic T lymphocytes. Enhanced cytosolic bioavailability of tumor-specific antigens improves access to human leukocyte antigen (HLA) class I molecules for more efficient cytotoxic T lymphocyte generation. Various cell-penetrating domains (CPDs) are known to ferry covalently linked heterologous antigens to the intracellular compartment by traversing the plasma membrane. OBJECTIVE: To determine whether generating melanoma antigen family A, 3 (MAGE-A3), a tumor-specific cancer-testis antigen, as a fusion protein with CPD will enhance the cytosolic bioavailability of MAGE-A3. DESIGN: MAGE-A3 was amplified by polymerase chain reaction using complementary DNA from renal tissue and cloned in frame with a CPD (YARKARRQARR) at the amino-terminal end and hexahistidine at the carboxy-terminal end to generate CPD-MAGE-A3 in a pQE-70 expression vector. Cultures were grown in Escherichia coli BL21 Star (DE3-pLysS) cells followed by nickel-nitrilotriacetic acid affinity purification of recombinant proteins. MAIN OUTCOMES AND MEASURES: Measurement of DC membrane penetration of CPD-MAGE-A3 vs MAGE-A3 and determination of the effect of CPD-MAGE-A3 pulsing on DC phenotypic expression of cell-surface antigens. RESULTS: Media composition and isopropyl-d-thiogalactosidase induction were optimized to achieve high levels of protein expression followed by purification. Western blot analysis with MAGE-A3 antibodies recognized both MAGE-A3 and CPD-MAGE-A3 proteins, while CPD antibodies recognized only CPD-MAGE-A3. Purified CPD-MAGE-A3 exhibited more efficient DC membrane penetration than did MAGE-A3 alone as confirmed by immunofluorescence analysis. High-level expression of several unique DC markers (CD80, CD83, CD86, and HLA-DR) by flow cytometry was consistent with a mature DC phenotype, indicating that pulsing with CPD-MAGE-A3 did not alter specific cell-surface antigens required for T-cell activation. CONCLUSIONS AND RELEVANCE: We have demonstrated for the first time, to our knowledge, that cloning and purification of MAGE-A3 with CPD enhances its cytosolic bioavailability in DCs without altering cell-surface antigens, potentially making it a more potent therapeutic cancer vaccine compared with existing MAGE-A3 protein and peptide vaccines.
Asunto(s)
Antígenos de Neoplasias/uso terapéutico , Vacunas contra el Cáncer/farmacocinética , Vacunas contra el Cáncer/uso terapéutico , Permeabilidad de la Membrana Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Proteínas de Neoplasias/farmacocinética , Proteínas de Neoplasias/uso terapéutico , Antígenos de Neoplasias/inmunología , Disponibilidad Biológica , Vacunas contra el Cáncer/inmunología , Permeabilidad de la Membrana Celular/inmunología , Péptidos de Penetración Celular , Clonación Molecular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Recent observations suggest a lower incidence of malignancies in patients infected with HIV during treatment with Highly Active Anti-Retroviral Therapy (HAART) utilizing protease inhibitors. We investigated the effects of ritonavir, a FDA approved HIV protease inhibitor, on proliferation of pancreatic ductal adeno-carcinoma (PDAC) cell lines. Human PDAC cell lines BxPC-3, MIA PaCa-2, and PANC-1 were propagated under standard conditions and treated with serial dilutions of ritonavir. Ritonavir inhibited cell growth in a dose-dependent manner as well as activated the intrinsic apoptotic pathway in human pancreatic ductal adenocarcinoma (PDAC) cell lines. We observed down-modulation of cell-cycle promoting and up-regulation of cell-cycle inhibitory genes; enhanced interaction of retinoblastoma protein (RB) with E2F-1 transcription factor; inhibition of phosphorylation of RB, resulting in sequestration of E2F-1 and subsequent down-regulation of S phase genes; decreased interaction of E2F-1 with its consensus binding sites; inhibition of cell motility and invasiveness; and inhibition of the AKT pathway. Our results demonstrate a potential use of ritonavir as part of combination chemotherapy for PDAC. Since ritonavir is FDA approved for HIV, drug repositioning for PDAC would limit the costs and reduce risks.
RESUMEN
MAGE-A3 is highly expressed in epithelial ovarian cancer (EOC), making it a promising candidate for immunotherapy. We investigated whether dendritic cells (DCs) transduced with a rAAV-6 capsid mutant vector Y445F could elicit effective MAGE-A3-specific anti-tumor cytotoxic T lymphocyte (CTL) responses in vitro. MAGE-A3 was cloned and rAAV-6-MAGE-A3 purified, followed by proviral genome detection using real-time PCR. Immunofluorescence detection of rAAV-6-Y445F-MAGE-A3-transduced DCs demonstrated 60% transduction efficiency. Fluorescent in situ hybridization analysis confirmed chromosomal integration of rAAV vectors. Flow cytometric analysis of transduced DCs showed unaltered expression of critical monocyte-derived surface molecules with retention of allo-stimulatory activity. Co-culture of autologous T lymphocytes with MAGE-A3-expressing DCs produced CTLs that secreted IFN-γ, and efficiently killed MAGE-A3+ EOC cells. This form of rAAV-based DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for management of EOC.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoterapia , Proteínas de Neoplasias/inmunología , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Linfocitos T Citotóxicos/inmunología , Cápside , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Células Dendríticas/citología , Células Dendríticas/inmunología , Dependovirus/genética , Vectores Genéticos , Humanos , Interferón gamma/inmunología , Prueba de Cultivo Mixto de Linfocitos , Mutación , Transducción GenéticaRESUMEN
PURPOSE: Enhancer of zeste homologue 2 (EZH2), a component of the chromatin modification protein complex, is upregulated in pancreatic ductal adenocarcinoma (PDAC), whereas loss of p53 and its downstream target, p21(waf1/cip1), is also observed frequently. We sought to investigate the role of the p53-p21(waf1/cip1) pathway in relation to EZH2-mediated inhibition of PDAC. METHODS: The PANC-1 cell line was utilized in chromatin immunoprecipitation, gene profiling, Western blot, cell invasion, cell proliferation, and tumor xenograft assays. RESULTS: Western blot analysis with antibodies that recognize both wild-type and mutant p53 did not show any alterations in band intensity; however, antibody that detects only mutant p53 showed a band of significantly lesser intensity with EZH2 knockdown. Western blot analysis further revealed a significant upregulation of p21(waf1/cip1). Gene expression profile analysis indicated significantly enhanced transcripts of transcriptional inducers of p21(waf1/cip1), with downregulation of mutant p53 transcript, corroborating the Western blot analysis. PANC-1 cells expressing EZH2-short hairpin RNA displayed markedly attenuated growth in SCID mice. CONCLUSION: Downregulation of mutant p53 with concomitant enhanced expression of p21(waf1/cip1) and its transcriptional trans-activators may contribute toward EZH2-mediated suppression of PDAC.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Genes p53/fisiología , Neoplasias Pancreáticas/genética , Complejo Represivo Polycomb 2/fisiología , ARN Interferente Pequeño/genética , Adenocarcinoma/patología , Animales , Apoptosis , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Elementos de Facilitación Genéticos , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Ratones , Neoplasias Pancreáticas/patología , Complejo Represivo Polycomb 2/genética , Regulación hacia ArribaRESUMEN
The enhancer of zeste homolog 2 (EZH2) methyltransferase is a transcriptional repressor. EZH2 is abnormally elevated in epithelial ovarian cancer (EOC). We demonstrated that EZH2 knockdown inhibited cell growth, activated apoptosis, and enhanced chemosensitivity. Further, silencing of EZH2 resulted in re-expression of p21(waf1/cip1) and down-regulation of mutant p53. Finally, EZH2 knockdown contributed to attenuated EOC growth in SCID mice.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/patología , Complejo Represivo Polycomb 2/metabolismo , Interferencia de ARN , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Humanos , Ratones , Ratones SCID , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Complejo Represivo Polycomb 2/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: To investigate the possibility of inhibiting the progression of pancreatic ductal adenocarcinoma (PDAC) by facilitating the expression of E-cadherin through the enforced expression of microRNA-101 (miR-101). METHODS: In situ hybridization was conducted with archival tissue using a double digoxigenin-labeled probe. Chromatin immunoprecipitation (ChIP) assay was conducted with EZ-Magna ChIPTM A. Gene profile analysis, Western blot, and immunoprecipitation assays were performed using standard protocols. RESULTS: We found that decreased miR-101 expression observed in archival patient tissues was significantly associated with poor prognosis indicated by low-intensity staining in high-grade tumors. ChIP assays using anti-enhancer of zeste homolog 2 (EZH2) antibodies indicated not only the interaction of EZH2 to the CDH1 (E-cadherin) promoter, but also that this interaction was significantly diminished in cells transfected with pre-miR-101. We observed a global downregulation of trimethylated lysine 27 of H3 histone (H3K27me3) along with upregulation of the enzymes histone deacetylase -1 and -2 with the re-expression of miR-101. Further, we observed lesser levels of transcriptional factors that inhibit the CDH1 promoter with pre-miR-101 treatment. Western blot analysis confirmed the enhanced E-cadherin expression. PANC-1 cells transduced with pre-miR-101 displayed markedly attenuated growth in SCID mice. CONCLUSION: These results suggest the potential therapeutic use of miR-101-enforced expression for inhibition of PDAC.
Asunto(s)
Cadherinas/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , MicroARNs/uso terapéutico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Adulto , Anciano , Animales , Antígenos CD , Apoptosis/genética , Secuencia de Bases , Carcinoma Ductal Pancreático/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Femenino , Expresión Génica , Humanos , Hibridación in Situ , Masculino , Ratones , Ratones SCID , MicroARNs/genética , Persona de Mediana Edad , Invasividad Neoplásica/genética , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Transducción Genética , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: MicroRNA-101 (miR-101) expression is negatively associated with tumor growth and proliferation in several solid epithelial cancers. Enhancer of zeste homolog 2 (EzH2) appears to be a functional target of miR-101. We explore the role of miR-101 and its interaction with EzH2 in epithelial ovarian carcinoma (EOC). METHODS: In situ hybridization (ISH) for miR-101 was performed on EOC patient tissues and normal controls. EOC cell lines were transfected with miR-101 and subjected to growth analysis and clonogenic assays. Cell motility was assessed by Boyden chamber and wound-healing assays. P21(waf1/cip1) and EzH2 interaction was assessed by Chromatin Immunoprecipitation (ChIP) assay in MDAH-2774 cells. SCID mice were assessed for tumor burden after injection with miR-101 or control vector-treated MDAH-2774 cells. RESULTS: ISH analysis revealed a decrease in miR-101 expression in EOC compared with normal tissue. MiR-101 re-expression in EOC cell lines resulted in increased apoptosis, decreased cellular proliferation, invasiveness, and reduced growth of tumor xenografts. CHIP assays revealed that re-expression of miR-101 inhibited the interaction of EzH2 with p21(waf1/cip1) promoter. CONCLUSIONS: MiR-101 re-expression appears to have antitumor effects, providing a better understanding of the role of miR-101 in EOC.
Asunto(s)
Cromatina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/genética , MicroARNs/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Factores de Transcripción/genética , Animales , Apoptosis , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular , Cromatina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones SCID , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Ovario/metabolismo , Ovario/patología , Complejo Represivo Polycomb 2 , Factores de Transcripción/metabolismoRESUMEN
Pure populations of tumor cells are essential for the identification of tumor-associated proteins for the development of targeted therapy. In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. The polycomb group (PcG) protein, enhancer of zeste homolog 2 (EzH2), a methyl-transferase that plays a key role in -transcriptional gene repression, is frequently overexpressed in several malignant tumors. High levels of EzH2 are often associated with advanced disease stage in many solid tumors; however, its role in the pathogenesis of pancreatic ductal adeno-carcinoma (PDAC) is poorly understood. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteomics studies largely depends on highly sensitive protein detection methods. Here, we developed a faster and sensitive Western blot protocol and validated it for the detection of EzH2 in â¼2,000 cells. Initially, cultured PANC-1 cells were used to optimize protein electrophoresis and western blotting conditions. Gradient gel electrophoresis in combination with optimized antibody concentrations, and a sensitive chemiluminescent assay provided a strong signal. In order to further confirm the role of EzH2 in PDAC, employing siRNA-mediated gene silencing via long lasting plasmid vectors containing shRNA, we investigated the potential role of EzH2 gene silencing in pancreatic cancer regression. Positive correlation of EzH2 expression was observed with advanced stage, serous histology, and increasing grade in pancreatic cancer patient tissues. Further EzH2 knockdown resulted in decreased cell growth and invasiveness. The findings of this study emphasize that western blotting of a LCM-generated pure population of cancer cells may be a valuable technique for the study of tumor-specific proteins.
Asunto(s)
Western Blotting/métodos , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Unión al ADN/metabolismo , Rayos Láser , Microdisección/métodos , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción/metabolismo , Animales , Carcinoma Ductal Pancreático/patología , Ensayos de Migración Celular/métodos , Proliferación Celular , Separación Celular/métodos , Criopreservación , Electroforesis en Gel de Poliacrilamida/métodos , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Microtomía/métodos , Neoplasias Pancreáticas/patología , Complejo Represivo Polycomb 2 , Ratas , Coloración y Etiquetado/métodos , Células Tumorales CultivadasRESUMEN
INTRODUCTION: The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. METHODS: BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was evaluated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To evaluate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS: SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. CONCLUSIONS: These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.
RESUMEN
BACKGROUND: Sulforaphane (SFN), an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC). The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC. RESULTS: SFN at concentrations of 5-20 microM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 microM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 microM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose)-polymerase (PARP). Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB) and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex. CONCLUSIONS: SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB) phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Factor de Transcripción E2F1/metabolismo , Células Epiteliales/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteína de Retinoblastoma/metabolismo , Tiocianatos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isotiocianatos , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Paclitaxel/farmacología , Fosforilación/efectos de los fármacos , Fase S/efectos de los fármacos , Sulfóxidos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
PURPOSE: The goal of this study was to investigate the effects of silencing HIF-1 alpha gene expression with specific small interfering RNA (siRNA) on VEGF production and angiogenesis in epithelial ovarian cancer (EOC) cells. METHODS: Two EOC cell lines, MDAH-2774 and SKOV-3, were cultured under normoxic (20% O(2)) and hypoxic (2% O(2)) conditions using standard techniques. After EOC cells were transfected with siRNA, HIF-1 alpha and VEGF mRNA levels were measured by real-time RT-PCR. Angiogenesis was evaluated utilizing an in vitro assay model consisting of human umbilical vein endothelial cells (HUVEC) and polymerized ECM Matrix. RESULTS: Both EOC cell lines evaluated constitutively expressed HIF-1 alpha and VEGF mRNA. HIF-1 alpha and VEGF mRNA levels were significantly increased in response to hypoxia (P < 0.05). Under hypoxic conditions, inhibition of HIF-1 alpha gene expression by a specific siRNA resulted in a significant reduction in HIF-1 alpha and VEGF mRNA levels (P < 0.05). In the in vitro angiogenesis model, supernatant from the hypoxic EOC cells induced the HUVEC to form a complex tubular network, a hallmark of angiogenesis. Semi-quantitative analysis of the angiogenesis assay revealed a significant reduction in tube formation when supernatant from HIF-1 alpha siRNA-treated hypoxic EOC cell was used (P < 0.05). CONCLUSION: Inhibition of HIF-1 alpha expression by specific siRNA resulted in a significant decrease in VEGF production and angiogenesis. Further investigation of HIF-1 alpha inhibition for anti-tumor activity is warranted and may potentially prove HIF-1 alpha as a therapeutic target in the management ovarian cancer.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Ováricas/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacologíaRESUMEN
BACKGROUND: Ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stages. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the 5-year survival rate. Chemotherapy with paclitaxel after surgery increases median survival only by 2 to 3 years in stage IV disease highlights the need for more effective drugs. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening of immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail treatment known as HAART (Highly Active Anti-Retroviral Therapy). RESULTS: Here we show that ritonavir, a HIV protease inhibitor effectively induced cell cycle arrest and apoptosis in ovarian cell lines MDH-2774 and SKOV-3 in a dose dependent manner. Over a 3 day period with 20 muM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line. Ritonavir caused G1 cell cycle arrest of the ovarian cancer cells, mediated by down modulating levels of RB phosphorylation and depleting the G1 cyclins, cyclin-dependent kinase and increasing their inhibitors as determined by gene profile analysis. Interestingly, the treatment of ritonavir decreased the amount of phosphorylated AKT in a dose-dependent manner. Furthermore, inhibition of AKT by specific siRNA synergistically increased the efficacy of the ritonavir-induced apoptosis. These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir. CONCLUSION: Our results demonstrate a potential use of ritonavir for ovarian cancer with additive effects in conjunction with conventional chemotherapeutic regimens. Since ritonavir is clinically approved for human use for HIV, drug repositioning for ovarian cancer could accelerate the process of traditional drug development. This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Ritonavir/farmacología , Biomarcadores de Tumor/metabolismo , Western Blotting , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Fosforilación/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/farmacología , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Cicatrización de Heridas/efectos de los fármacosRESUMEN
A prominent feature of most if not all cancers is a striking genetic instability, leading to ongoing accrual of mutational changes, some of which underlie tumor progression, including acquisition of invasiveness, drug resistance, and metastasis. Thus, the molecular basis for the generation of this genetic diversity in cancer cells has important implications in understanding cancer progression. Here we report that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to an increased rate of mutation and progressive accumulation of genetic variation over time. We demonstrate that the inhibition of HR activity in MM cells by small inhibitory RNA (siRNAs) targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome and, conversely, the induction of HR activity leads to significant elevation in the number of new mutations over time and development of drug resistance in MM cells. These data identify dysregulated HR activity as a key mediator of DNA instability and progression of MM, with potential as a therapeutic target.
Asunto(s)
Perfilación de la Expresión Génica , Inestabilidad Genómica , Mieloma Múltiple/genética , Recombinación Genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Pérdida de Heterocigocidad , Mutación , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , TransfecciónRESUMEN
PURPOSE: The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. RESULTS: Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. CONCLUSIONS: We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.