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1.
Adv Exp Med Biol ; 1290: 51-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33559854

RESUMO

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.


Assuntos
Interleucina-10 , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Células Dendríticas , Feminino , Humanos , Neoplasias Ovarianas/terapia , Transdução de Sinais , Microambiente Tumoral
2.
Blood ; 113(10): 2290-7, 2009 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-19050310

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica , Instabilidade Genômica , Mieloma Múltiplo/genética , Recombinação Genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Mutação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Transfecção
3.
Pharm Res ; 28(12): 3079-90, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21818714

RESUMO

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.


Assuntos
Cromatina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fatores de Transcrição/genética , Animais , Apoptose , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Ovário/patologia , Complexo Repressor Polycomb 2 , Fatores de Transcrição/metabolismo
4.
Int J Gynecol Cancer ; 21(2): 257-62, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21475076

RESUMO

OBJECTIVE: To compare the survival of patients with bilateral versus unilateral malignant ovarian germ cell tumors (OGCT). METHODS: Patients with a diagnosis of OGCT were identified from the Surveillance, Epidemiology, and End Results Program for the period 1988 to 2006 and were divided into bilateral and unilateral subgroups. Only surgically treated patients were included. Histologic types were grouped into dysgerminoma, malignant teratoma, and mixed germ cell tumors with pure nondysgerminoma cell tumors. Statistical analysis using Wilcoxon rank sum test, Kaplan-Meier survival methods, and Cox proportional hazards regression model were performed. RESULTS: In 1529 patients with OGCT, 1463 (95.7%) were unilateral and 66 (4.3%) were bilateral. Bilaterality was more common with dysgerminomas (6.5%) and mixed germ cell tumors with pure nondysgerminoma cell tumors (6.25%) than with immature teratomas (1.7%), P < 0.001. Most OGCT (67.3%) were stage I. Bilateral OGCT were more likely than unilateral tumors to be associated with advanced-stage disease (FIGO III and IV, 41% vs 20%, P < 0.04). Overall 5-year survival was 93.6% for unilateral OGCT and 80.7% in bilateral OGCT, P < 0.001. In multivariate analysis, bilaterality was not an independent predictor of survival when controlling for age, histology, stage, and surgical staging (hazard ratio, 1.3; 95% confidence interval, 0.7-2.5; P = 0.40). CONCLUSIONS: Compared with unilateral tumors, bilateral OGCT are more often associated with advanced-stage disease, high-risk histology, and poor survival. When other prognostic factors are accounted for, bilaterality was not an independent prognostic predictor of survival.


Assuntos
Neoplasias Embrionárias de Células Germinativas/mortalidade , Neoplasias Ovarianas/mortalidade , Adulto , Feminino , Humanos , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Embrionárias de Células Germinativas/cirurgia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgia , Prognóstico , Programa de SEER , Análise de Sobrevida , Estados Unidos , Adulto Jovem
5.
Mol Cancer ; 9: 47, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20196847

RESUMO

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.


Assuntos
Ciclo Celular/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Células Epiteliais/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína do Retinoblastoma/metabolismo , Tiocianatos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isotiocianatos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Fase S/efeitos dos fármacos , Sulfóxidos , Cicatrização/efeitos dos fármacos
6.
Arch Gynecol Obstet ; 282(6): 677-83, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20140681

RESUMO

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.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Ovarianas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia
7.
Mol Cancer ; 8: 26, 2009 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-19386116

RESUMO

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.


Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ritonavir/farmacologia , Biomarcadores Tumorais/metabolismo , Western Blotting , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/farmacologia , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Cicatrização/efeitos dos fármacos
8.
Clin Cancer Res ; 14(15): 4971-80, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18676772

RESUMO

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.


Assuntos
Adenocarcinoma/ultraestrutura , Esôfago de Barrett/ultraestrutura , Telomerase/antagonistas & inibidores , Telômero/ultraestrutura , Adenocarcinoma/metabolismo , Animais , Apoptose , Esôfago de Barrett/metabolismo , Linhagem Celular Tumoral , Senescência Celular , Células Epiteliais/ultraestrutura , Feminino , Humanos , Lasers , Camundongos , Camundongos SCID , Microdissecção , Transplante de Neoplasias , Telomerase/metabolismo
9.
Surgery ; 166(4): 503-508, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31416604

RESUMO

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.


Assuntos
Morte Celular/imunologia , Proteínas Ligadas por GPI/farmacologia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Neoplasias Pancreáticas/patologia , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular Tumoral , Eletroporação/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Células Matadoras Naturais/efeitos dos fármacos , Mesotelina , Neoplasias Pancreáticas/terapia , Sensibilidade e Especificidade , Microambiente Tumoral
10.
Surgery ; 163(3): 627-632, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29336814

RESUMO

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.


Assuntos
Proteínas Ligadas por GPI/farmacologia , Interleucina-10/fisiologia , Neoplasias Pancreáticas/patologia , Linfócitos T/fisiologia , Microambiente Tumoral/fisiologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Imunoterapia , Mesotelina , Receptores de Antígenos de Linfócitos T
11.
Cancer Res ; 65(21): 10041-9, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16267030

RESUMO

Myeloma vaccines, based on dendritic cells pulsed with idiotype or tumor lysate, have been met with limited success, probably in part due to insufficient cross-priming of myeloma antigens. A powerful method to introduce myeloma-associated antigens into the cytosol of dendritic cells is protein transduction, a process by which proteins fused with a protein transduction domain (PTD) freely traverse membrane barriers. NY-ESO-1, an immunogenic antigen by itself highly expressed in 60% of high-risk myeloma patients, was purified to near homogeneity both alone and as a recombinant fusion protein with a PTD, derived from HIV-Tat. Efficient entry of PTD-NY-ESO-1 into dendritic cells, confirmed by microscopy, Western blotting, and intracellular flow cytometry, was achieved without affecting dendritic cell phenotype. Experiments with amiloride, which inhibits endocytosis, and N-acetyl-l-leucinyl-l-norleucinal, a proteasome inhibitor, confirmed that PTD-NY-ESO-1 entered dendritic cells by protein transduction and was degraded by the proteasome. Tetramer analysis indicated superior generation of HLA-A2.1, CD8+ T lymphocytes specific for NY-ESO-1(157-165) with PTD-NY-ESO-1 compared with NY-ESO-1 control protein (44% versus 2%, respectively). NY-ESO-1-specific T lymphocytes generated with PTD-NY-ESO-1 secreted IFN-gamma indicative of a Tc1-type cytokine response. Thus, PTD-NY-ESO-1 accesses the cytoplasm by protein transduction, is processed by the proteasome, and NY-ESO-1 peptides presented by HLA class I elicit NY-ESO-1-specific T lymphocytes.


Assuntos
Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Proteínas de Membrana/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Amilorida/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Células Dendríticas/metabolismo , Fibroblastos/fisiologia , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Monócitos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T/imunologia , Transdução Genética
12.
Cancer Res ; 62(10): 2982-5, 2002 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12019182

RESUMO

Adeno-associated virus type 2 (AAV) is known to inhibit virally mediated oncogenic transformation. One of the early events of adenovirus (Ad) infection is the functional inactivation of cell cycle regulatory retinoblastoma (RB) family of proteins, which consists of retinoblastoma protein (pRB), p107, and p130. In an effort to understand the molecular basis of anti-oncogenic properties of AAV, we studied the effects of AAV expression on these proteins in cells infected with Ad. Western blot analysis showed that AAV interferes with the adenoviral-induced degradation and hyperphosphorylation of the pRB family of proteins in normal human fibroblasts as well as in HeLa and 293 cell lines. RNase protection assay showed enhanced expression of pocket protein gene by AAV expression. We also demonstrate that Rep proteins, the major AAV regulatory proteins, bind to E1A, the immediate early gene of Ad responsible for hyperphosphorylation and dissociation of pRB-E2F complex. This binding of AAV Rep proteins to E1A leads to decreased association between E1A and pRB leading to protection of pocket proteins from degradation, decreased expression of S phase genes and inhibition of cell cycle progression. These results suggest that the antiproliferative activity of AAV against Ad is mediated, at least in part, by effects of AAV Rep proteins on the Rb family of proteins.


Assuntos
Dependovirus/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Células HeLa , Humanos , Ligação Proteica , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/fisiologia
13.
Mol Cancer ; 4: 24, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16022731

RESUMO

BACKGROUND: In cancer cells, telomerase induction helps maintain telomere length and thereby bypasses senescence and provides enhanced replicative potential. Chemical inhibitors of telomerase have been shown to reactivate telomere shortening and cause replicative senescence and apoptotic cell death of tumor cells while having little or no effect on normal diploid cells. RESULTS: We designed siRNAs against two different regions of telomerase gene and evaluated their effect on telomere length, proliferative potential, and gene expression in Barrett's adenocarcinoma SEG-1 cells. The mixture of siRNAs in nanomolar concentrations caused a loss of telomerase activity that appeared as early as day 1 and was essentially complete at day 3. Inhibition of telomerase activity was associated with marked reduction in median telomere length and complete loss of detectable telomeres in more than 50% of the treated cells. Telomere loss caused senescence in 40% and apoptosis in 86% of the treated cells. These responses appeared to be associated with activation of DNA sensor HR23B and subsequent activation of p53 homolog p73 and p63 and E2F1. Changes in these gene regulators were probably the source of observed up-regulation of cell cycle inhibitors, p16 and GADD45. Elevated transcript levels of FasL, Fas and caspase 8 that activate death receptors and CARD 9 that interacts with Bcl10 and NFKB to enhance mitochondrial translocation and activation of caspase 9 were also observed. CONCLUSION: These studies show that telomerase siRNAs can cause effective suppression of telomerase and telomere shortening leading to both cell cycle arrest and apoptosis via mechanisms that include up-regulation of several genes involved in cell cycle arrest and apoptosis. Telomerase siRNAs may therefore be strong candidates for highly selective therapy for chemoprevention and treatment of Barrett's adenocarcinoma.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose , Senescência Celular/fisiologia , Telomerase/genética , Telomerase/metabolismo , Adenocarcinoma/genética , Linhagem Celular , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , RNA Interferente Pequeno/genética , Telômero/genética
14.
Surgery ; 158(4): 981-6; discussion 986-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26189069

RESUMO

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.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células/fisiologia , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Células-Tronco Neoplásicas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Transfecção , Proteína Supressora de Tumor p53/genética
15.
Leuk Res ; 27(1): 73-8, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12479855

RESUMO

Multiple myeloma is a malignant proliferation of plasma cells which fail to undergo apoptosis. To understand events associated with lack of apoptosis in these cells, we studied effect of antisense p53 gene transduction in a multiple myeloma cell line, ARH77. Adeno-associated virus was used as a vector to introduce p53 cDNA in an antisense orientation driven by a herpes virus thymidine kinase promoter. We observed, that an antisense p53 (p53as) transduced cell line showed marked reduction in p53 mRNA and protein expression and increased growth when compared to the control cell lines transduced with neomycin-resistance gene or untransduced cells. There was a concomitant up-regulation of bcl-2 expression by over five-fold in p53as-transduced cells compared with controls; while there was no significant change in expression of c-myc and IL-6, genes implicated in myeloma growth. We measured apoptosis in the transduced cells by DNA end-labeling reaction which revealed decrease in apoptosis from 15.6% in control cells to 1.6% in p53as-transduced cells. Additionally, the p53as cells over expressing bcl-2 also showed resistance to killing by dexamethasone. In summary, our data demonstrates that loss of p53 function leads to myeloma cell progression and resistant phenotype through bcl-2-related mechanisms.


Assuntos
Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Genes p53 , Mieloma Múltiplo/genética , Proteínas de Neoplasias/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína Supressora de Tumor p53/fisiologia , Apoptose/genética , Divisão Celular/efeitos dos fármacos , DNA Complementar/genética , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Proteína Supressora de Tumor p53/deficiência
16.
JAMA Surg ; 149(5): 451-7, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24671426

RESUMO

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.


Assuntos
Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/farmacocinética , Vacinas Anticâncer/uso terapêutico , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Proteínas de Neoplasias/farmacocinética , Proteínas de Neoplasias/uso terapêutico , Antígenos de Neoplasias/imunologia , Disponibilidade Biológica , Vacinas Anticâncer/imunologia , Permeabilidade da Membrana Celular/imunologia , Peptídeos Penetradores de Células , Clonagem Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
17.
Pharmaceuticals (Basel) ; 7(1): 46-57, 2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24451403

RESUMO

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.

18.
Vaccine ; 32(8): 938-43, 2014 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-24406390

RESUMO

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.


Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia , Proteínas de Neoplasias/imunologia , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Linfócitos T Citotóxicos/imunologia , Capsídeo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/imunologia , Dependovirus/genética , Vetores Genéticos , Humanos , Interferon gama/imunologia , Teste de Cultura Mista de Linfócitos , Mutação , Transdução Genética
19.
Surgery ; 154(4): 739-46; discussion 746-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24074410

RESUMO

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.


Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Genes p53/fisiologia , Neoplasias Pancreáticas/genética , Complexo Repressor Polycomb 2/fisiologia , RNA Interferente Pequeno/genética , Adenocarcinoma/patologia , Animais , Apoptose , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Elementos Facilitadores Genéticos , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Camundongos , Neoplasias Pancreáticas/patologia , Complexo Repressor Polycomb 2/genética , Regulação para Cima
20.
Cancer Lett ; 336(1): 53-60, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23603558

RESUMO

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.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/patologia , Complexo Repressor Polycomb 2/metabolismo , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Humanos , Camundongos , Camundongos SCID , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Complexo Repressor Polycomb 2/genética , Proteína Supressora de Tumor p53/genética
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