RESUMO
Hepatocellular carcinoma (HCC) is the third leading form of cancer worldwide, and its incidence is increasing rapidly in the United States, tripling over the past 3 decades. The current chemotherapeutic strategies against localized and metastatic HCC are ineffective. Here we report that 6-methoxyethylamino-numonafide (MEAN) is a potent growth inhibitor of murine xenografts of 2 human HCC cell lines. At the same dose and with the same treatment strategies, MEAN was more efficacious in inhibiting tumor growth in mice than sorafenib, the only approved drug for HCC. Treatment by MEAN at an effective dose for 6 wk was well tolerated by animals. Combined therapy using both sorafenib and MEAN enhanced tumor growth inhibition over monotherapy with either agent. Additional experiments revealed that MEAN inhibited tumor growth through mechanisms distinct from those of either its parent compound, amonafide, or sorafenib. MEAN suppressed C-MYC expression and increased expression of several tumor suppressor genes, including Src homology region 2 domain-containing phosphatase-1 (SHP-1) and TXNIP (thioredoxin-interacting protein). As an encouraging feature for envisioned clinical application, the IC50 of MEAN was not significantly changed in several drug-resistant cell lines with activated P-glycoprotein drug efflux pumps compared to drug-sensitive parent cells, demonstrating the ability of MEAN to be effective in cells resistant to existing chemotherapy regimens. MEAN is a promising candidate for clinical development as a single-agent therapy or in combination with sorafenib for the management of HCC.-Liu, Y., Lou, G., Norton, J. T., Wang, C., Kandela, I., Tang, S., Shank, N. I., Gupta, P., Huang, M., Avram, M. J., Green, R., Mazar, A., Appella, D., Chen, Z., Huang, S. 6-Methoxyethylamino-numonafide inhibits hepatocellular carcinoma xenograft growth as a single agent and in combination with sorafenib.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Naftalimidas/uso terapêutico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Alanina Transaminase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Niacinamida/uso terapêutico , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The perinucleolar compartment (PNC) is a nuclear substructure associated with, but structurally distinct from, the nucleolus. The PNC contains several RNA processing proteins and several RNA pol III transcripts, which form novel complexes. As determined by cell culture experiments and human tumor samples, the PNC forms exclusively in cancer cells and the percentage of cancer cells in a population that have one or more PNCs directly correlates with the malignancy of that population of cells. Therefore, the PNC is being developed as a prognostic marker for several malignancies. PNC elimination in cancer cells has proven to be a useful as screening method to discover probe compounds used to elucidate PNC biology and to discover compounds with the potential to be developed as minimally toxic anti-cancer drugs.
Assuntos
Nucléolo Celular , Núcleo Celular , Antineoplásicos , Humanos , Neoplasias , RNARESUMO
Nitrogen bisphosphonates (NBPs) are commonly prescribed for osteoporosis but have also been found to induce inflammatory reactions and to delay the progression of breast cancer. The inflammatory and anticancer effects of the NBPs might be associated with an ability to modulate innate immune signaling. In mice, intraperitoneal NBP administration causes a rapid influx of neutrophils and monocytes that is dependent on the myeloid differentiation primary response gene 88 (MyD88) mediator of Toll-like receptor (TLR) and IL-1 signaling. Bone marrow chimeras demonstrate that this inflammatory response is partially dependent on TLR4 expression by hematopoietic cells and the IL-1 receptor on radioresistant cells. In vitro, NBPs directly stimulate neither murine bone marrow-derived mononuclear cells nor human peripheral blood mononuclear cells, but rather prime them to produce increased amounts of cytokines when exposed to IL-1 or TLR ligands. This potentiation is mediated by a reduction in IL-1 receptor-associated kinase-M, a negative regulator of MyD88-dependent signaling. In vivo, this property renders the NBPs as effective adjuvants that enhance both cellular and antibody responses to antigens.
Assuntos
Difosfonatos/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/fisiologia , Inflamação/etiologia , Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Difosfonatos/toxicidade , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Peritonite/etiologia , Peritonite/imunologia , Peritonite/metabolismo , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/metabolismoRESUMO
Amonafide is a DNA intercalator in clinical development for the treatment of cancer. The drug has a 5-position amine that is variably acetylated to form a toxic metabolite in humans, increasing adverse effects and complicating the dosing of amonafide. Numonafides, 6-amino derivatives of amonafide that avoid the toxic acetylation, also show in vitro anticancer activity, as we have previously described. Here, we report the in vitro and in vivo activities of two numonafides, 6-methoxyethylamino-numonafide (MEAN) and 6-amino-numonafide (AN) with comparisons to amonafide. The in vitro potencies and cellular anticancer mechanisms are similar for the two numonafides and amonafide. Results from several mouse models of human cancer demonstrate that AN and MEAN require slightly higher doses than amonafide for equal efficacy in short-term dosing models, but the same dose of all three compounds in long-term dosing models are equally efficacious. MEAN is tolerated much better than amonafide and AN at equally efficacious doses based on weight change, activity, stool consistency, and dose tolerance with survival as the end point. The studies presented here demonstrate that MEAN is much less toxic than amonafide or AN in mouse models of human liver and gastric cancers while being equally efficacious in vivo and inhibiting cancer cells through similar mechanisms. These findings demonstrate that numonafides can be less toxic than amonafide and support further preclinical development and novel anticancer agents or as replacements or amonafide.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Naftalimidas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Adenina , Animais , Linhagem Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Camundongos , Camundongos Nus , Naftalimidas/efeitos adversos , Naftalimidas/química , Organofosfonatos , Neoplasias Gástricas/induzido quimicamente , Transplante HeterólogoRESUMO
There remains a compelling need for the development of treatments for unresectable melanoma. Agents that stimulate the innate immune response could provide advantages for cell-based therapies. However, there are conflicting reports concerning whether toll-like receptor (TLR) signaling controls tumor growth. The objective of this study was to evaluate the effect of intralesional administration of a TLR7 agonist in melanoma therapy. B16cOVA melanoma was implanted to TLR7 mice to evaluate the roles of stromal TLR7 on melanoma growth. To capitalize on the potential deleterious effects of TLR7 stimulation on the tumor growth, we injected melanoma tumor nodules with a newly developed and potent TLR7 agonist. B16 melanoma nodules expanded more rapidly in TLR7-deficient and MyD88 mice compared with TLR9 and wild type mice. Repeated injections with low doses of unconjugated TLR7 agonist were more effective at attenuating nodule size than a single high dose injection. To improve the efficacy we conjugated the agonist to phospholipid or phospholipids-polyethylene glycol, which retained TLR7 specificity. The phospholipid conjugate was indeed more effective in reducing lesion size. Furthermore, intralesional administration of the phospholipid TLR7 agonist conjugate enhanced the antimelanoma effects of systemic treatment with interleukin (IL)-2 and prolonged the survival of mice compared with IL-2 alone. Our study showed that: (1) TLR7/MyD88 signaling in the stroma is involved in melanoma growth; and (2) intralesional administration of a TLR7 agonist reduces the growth of melanoma nodules and enhances the antimelanoma effects of IL-2.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/farmacologia , Interleucina-2/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfolipídeos/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo , Adenina/farmacologia , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Sistema Imunitário , Interleucina-2/farmacologia , Melanoma/tratamento farmacológico , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Transplante de Neoplasias , Receptor Toll-Like 9/genéticaRESUMO
All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)-fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure.
Assuntos
Bioensaio/métodos , Núcleo Celular/ultraestrutura , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/patologia , Algoritmos , Antineoplásicos/uso terapêutico , Bioensaio/instrumentação , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Invasividade Neoplásica/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The perinucleolar compartment (PNC) is a nuclear subdomain that is unique to tumor cells, and the percentage of cells in a population containing PNCs (PNC prevalence) indicates the level of malignancy of that population. Here, we utilize anti-cancer drugs and other exogenous stimuli to investigate the structure and function of the PNC. Screening of clinically used anti-cancer drugs revealed two types of drugs disassemble PNCs and do so through their specific molecular actions. Transcription inhibitors reduce PNC prevalence in parallel with RNA polymerase III transcription reduction, and a subset of DNA-damaging drugs and stimuli (UV radiation) disassemble the PNC. Inhibition of cellular DNA damage response demonstrated that the DNA damage itself, not the response or polymerase III inhibition, is responsible for PNC disassembly, suggesting that the maintenance of the PNC is dependent upon DNA integrity. Analyses of the types of DNA damage that cause PNC disassembly show that interstrand DNA base pairing, not strand continuity, is important for PNC integrity, indicating that the PNC components are directly interacting with the DNA. Complementary cell biology experiments demonstrated that the number of PNCs per cell increases with the rounds of endoreplication and that PNCs split into doublets during mid S phase, both of which are phenotypes that are typical of a replicating DNA loci. Together, these studies validate PNC disassembly as a screening marker to identify chemical probes and revealed that the PNC is directly nucleated on a DNA locus, suggesting a potential role for the PNC in gene expression regulation.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Dano ao DNA , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , RNA Polimerase III/metabolismo , Transcrição Gênica , Células HeLa , Humanos , Neoplasias , Fase SRESUMO
BACKGROUND: The perinucleolar compartment (PNC) is a subnuclear structure localized at the nucleolar periphery. Previous studies using breast cancer as a model system demonstrated that PNC prevalence (the percentage of cells with 1 or more PNC) increased with disease progression and was associated with poor patient outcomes. METHODS: To evaluate the validity of developing PNC prevalence as a novel pancancer prognostic marker, the authors investigated whether PNC prevalence was correlated with malignancy in a spectrum of tissue types and evaluated its selective association with malignancy under various experimental conditions. RESULTS: PNC prevalence was low in primary and immortalized cells and in cell lines derived from hematologic malignancies, but it was heterogeneous in cell lines derived from solid tumors, including those of epithelial and nonepithelial origins. Studies using human myometrial tissue and thyroid cancer cell lines with various levels of malignancy demonstrated a correlation between high PNC prevalence and malignant potential. Furthermore, PNC prevalence corresponded directly to metastatic capacities in a series of well characterized cell lines of the same origin that were selected for various levels of metastatic capacity in a mouse model. Conversely, PNC prevalence was reduced experimentally by over expressing an antimetastatic protein in breast cancer cells. However, PNC prevalence was not associated with traits that were shared by both cancer and normal cells, including proliferation, glycolysis, and differentiation. CONCLUSIONS: Together, these observations helped to verify that PNC prevalence selectively represents malignancy in a broad spectrum of solid tissue tumors, demonstrating its potential to be developed as a pancancer prognostic marker of malignancy.
Assuntos
Biomarcadores , Nucléolo Celular , Corpos de Inclusão Intranuclear/ultraestrutura , Neoplasias/diagnóstico , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias/ultraestrutura , FenótipoRESUMO
RNA processing is altered during malignant transformation, and expression of the polypyrimidine tract-binding protein (PTB) is often increased in cancer cells. Although some data support that PTB promotes cancer, the functional contribution of PTB to the malignant phenotype remains to be clarified. Here we report that although PTB levels are generally increased in cancer cell lines from multiple origins and in endometrial adenocarcinoma tumors, there appears to be no correlation between PTB levels and disease severity or metastatic capacity. The three isoforms of PTB increase heterogeneously among different tumor cells. PTB knockdown in transformed cells by small interfering RNA decreases cellular growth in monolayer culture and to a greater extent in semi-solid media without inducing apoptosis. Down-regulation of PTB expression in a normal cell line reduces proliferation even more significantly. Reduction of PTB inhibits the invasive behavior of two cancer cell lines in Matrigel invasion assays but enhances the invasive behavior of another. At the molecular level, PTB in various cell lines differentially affects the alternative splicing pattern of the same substrates, such as caspase 2. Furthermore, overexpression of PTB does not enhance proliferation, anchorage-independent growth, or invasion in immortalized or normal cells. These data demonstrate that PTB is not oncogenic and can either promote or antagonize a malignant trait dependent upon the specific intra-cellular environment.
Assuntos
Neoplasias/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Apoptose , Caspase 2/metabolismo , Linhagem Celular , Transformação Celular Neoplásica , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/classificação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , Transcrição Gênica/genéticaRESUMO
Amonafide is a DNA intercalator and topoisomerase II inhibitor in clinical development for the treatment of neoplastic diseases. Amonafide contains a free arylamine, which causes it to be metabolized in humans by N-acetyl transferase-2 (NAT2) into a toxic form. To eliminate the NAT2 acetylation of amonafide while retaining the anticancer properties, we have synthesized nine derivatives that are structurally similar to amonafide that should not be acetylated. Eight derivatives have arylamines at the 6-position (vs. 5-position of amonafide) and one derivative completely lacks the arylamine. The derivative with a free amine in the 6-position and one with a substituted amine in the 6-position are not acetylated, whereas amonafide is extensively acetylated as determined by an NAT2 assay. The biological activities of these compounds were evaluated to determine whether they behaved similarly to amonafide in purified systems and in vitro. We found that three compounds had similar cancer cell-selective growth inhibition to amonafide, while retaining similar subcellular localization, DNA intercalation and topoisomerase II inhibition activities. In addition, these compounds were able to eliminate a marker of metastatic potential, the perinucleolar compartment. These three compounds (named numonafides) might thus allow for better patient management than those treated with amonafide; hence, they should be developed further as potential clinical replacements for amonafide or as novel anticancer drugs.