RESUMO
BACKGROUND: Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors. METHODS: RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors. RESULTS: Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFß1 and TGFß2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection. CONCLUSIONS: The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.
Assuntos
Carcinoma in Situ/genética , Cistadenocarcinoma Seroso/genética , Tubas Uterinas/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Ovarianas/genética , Biomarcadores Tumorais , Carcinoma in Situ/patologia , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/patologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Gradação de Tumores , Estadiamento de Neoplasias , Especificidade de Órgãos/genética , Neoplasias Ovarianas/patologia , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
We develop a random walk model to simulate the Brownian motion and the electrochemical response of a single molecule confined to an electrode surface via a flexible molecular tether. We use our simple model, which requires no prior knowledge of the physics of the molecular tether, to predict and better understand the voltammetric response of surface-confined redox molecules when motion of the redox molecule becomes important. The single molecule is confined to a hemispherical volume with a maximum radius determined by the flexible molecular tether (5-20 nm) and is allowed to undergo true three-dimensional diffusion. Distance- and potential-dependent electron transfer probabilities are evaluated throughout the simulations to generate cyclic voltammograms of the model system. We find that at sufficiently slow cyclic voltammetric scan rates the electrochemical reaction behaves like an adsorbed redox molecule with no mass transfer limitation; thus, the peak current is proportional to the scan rate. Conversely, at faster scan rates the diffusional motion of the molecule limits the simulated peak current, which exhibits a linear dependence on the square root of the scan rate. The switch between these two limiting regimes occurs when the diffusion layer thickness, (2Dt)(1/2), is ~10 times the tether length. Finally, we find that our model predicts the voltammetric behavior of a redox-active methylene blue tethered to an electrode surface via short flexible single-stranded, polythymine DNAs, allowing the estimation of diffusion coefficients for the end-tethered molecule.
Assuntos
Modelos Químicos , DNA/química , Difusão , Técnicas Eletroquímicas , Eletrodos , Azul de Metileno/química , Oxirredução , Propriedades de SuperfícieRESUMO
Stimulator of interferon genes (STING) is an innate immune receptor activated by natural or synthetic agonists to elicit antitumoral immune response via type I IFNs and other inflammatory cytokines. Bacillus Calmette-Guerin (BCG) is the standard of care as intravesical therapy for patients with high-risk non-muscle invasive bladder cancer (NMIBC). There are limited options available for patients with NMIBC who developed BCG unresponsiveness. In this study, we characterized in vitro and in vivo antitumor effects of E7766, a macrocyle-bridged STING agonist, via intravesical instillation in two syngeneic orthotopic murine NMIBC tumor models resistant to therapeutic doses of BCG and anti-PD-1 agents. E7766 bound to recombinant STING protein with a Kd value of 40 nmol/L and induced IFNß expression in primary human peripheral blood mononuclear cells harboring any of seven major STING genotypes with EC50 values of 0.15 to 0.79 µmol/L. Intravesical E7766 was efficacious in both NMIBC models with induction of effective immunologic memory in the treated animals. Pharmacologic activation of the STING pathway in the bladder resulted in IFN pathway activation, infiltration of T cells and natural killer (NK) cells, dendritic cell activation, and antigen presentation in bladder epithelium, leading to the antitumor activity and immunity. In addition, measurements of the pharmacodynamic markers, Ifnß1 and CXCL10, in bladder, urine, and plasma, and of STING pathway intactness in cancer cells, supported this mode of action. Taken together, our studies reveal an antitumor immune effect of pharmacologic activation of the STING pathway in bladder epithelium and thus provide a rationale for subsequent clinical studies in patients with NMIBC.
Assuntos
Fosfatidilinositol 3-Quinases , Neoplasias da Bexiga Urinária , Animais , Vacina BCG/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologiaRESUMO
A strategy for creating potent and pan-genotypic stimulator of interferon genes (STING) agonists is described. Locking a bioactive U-shaped conformation of cyclic dinucleotides by introducing a transannular macrocyclic bridge between the nucleic acid bases leads to a topologically novel macrocycle-bridged STING agonist (MBSA). In addition to substantially enhanced potency, the newly designed MBSAs, exemplified by clinical candidate E7766, exhibit broad pan-genotypic activity in all major human STING variants. E7766 is shown to have potent antitumor activity with long lasting immune memory response in a mouse liver metastatic tumor model. Two complementary stereoselective synthetic routes to E7766 are also described.
Assuntos
Antineoplásicos/farmacologia , Interferons/agonistas , Compostos Macrocíclicos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologiaRESUMO
Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piperidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazóis/farmacologia , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Hidrólise/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Piperidinas/química , Bibliotecas de Moléculas Pequenas/química , Tiazóis/químicaRESUMO
PURPOSE: We compared the efficacy and safety of a combined thulium laser incision and bipolar resection of prostate technique (web procedure) with traditional bipolar TURP. MATERIALS AND METHODS: We reviewed the medical records of 96 web procedure, 93 traditional bipolar TURP patients between 2013 and 2016. The web procedure consisted of thulium laser incision of the prostate at 3, 5, 7, 9 and 12 o'clock positions up to the resection plane and subsequent bipolar resection of the created prostate blocks. Resected tissue weight, operative time, resection velocity, complications, blood loss, and early operative outcome were compared. RESULTS: No significant differences were noted among the web procedure (web group) and traditional bipolar TURP in preoperative PSA ( 6.3 vs 8.7 ng/mL, P =0.295), preoperative postvoid residual urine (55.1 vs 76.4, P =.056), modified hemoglobin decrease (defined as total Hb decrease divided by the weight of the resected tissue: 0.060 vs 0.051, p=.380), complication rate (5.2% vs 5.3 %, P =.958), hospitalization (4.0 vs 4.2 days, P =.120) and catheterization (2.5 vs 3.4, p=.066). The resection velocity was higher in the web group (0.23 vs 0.17 g/ min, p=.001). In subgroup analysis, the significant difference of resection velocity between two group was showed in large prostates (>40 g: 0.25 vs 0.20 g/min, P =0.02 ) but not in the small prostate group. There was no difference in postoperative postvoid residual urine (21.9 vs 30.3 P =.231) and postvoid residual urine decrease (33.1 vs 45.5, P = .167) 2 months after surgery. CONCLUSION: The combination thulium laser incision and bipolar TURP technique had a higher resection efficiency and comparable efficacy and safety than traditional bipolar TURP.
Assuntos
Eletrocirurgia , Terapia a Laser , Prostatectomia , Hiperplasia Prostática/cirurgia , Túlio , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Humanos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Hiperplasia Prostática/patologia , Estudos RetrospectivosRESUMO
BACKGROUND: It was previously thought that renal hilum en bloc ligation during kidney removal may increase the risk of renal arteriovenous fistula (AVF) formation. We evaluated the safety and effectiveness of en bloc ligation of the renal pedicle using a stapler during laparoscopic nephrectomy and nephroureterectomy. METHODS: Ninety patients underwent en bloc renal hilar ligation using 45-mm endo-gastrointestinal anastomosis stapler and 2.5-mm staples during laparoscopic nephrectomy and nephroureterectomy from 2002 to 2015. Perioperative outcomes including estimated blood loss, operative time, and perioperative complications were documented. RESULTS: Twenty-seven patients underwent nephrectomy and 63 patients underwent nephroureterectomy. Thirty patients (33.3%) had perioperative complications. The majority of complications were of Clavien grade II. Six patients had Clavien Dindo grade III and none of the patients had grade IV complications. There were no significant differences in complication rates between right- and left-sided procedures (p = 0.233). Median blood loss was 190.1 ml and mean operative time was 227.1 minutes in nephrectomy and 256.6 minutes in nephroureterectomy. None of the patients had evidence of AVF formation on postoperative computerized tomography scan. Only three patients had a new diagnosis of heart failure. One of them was eventually lost to follow up. The remaining two patients had no evidence of AVF formation in imaging studies and physical examination. CONCLUSION: En bloc ligation of the renal pedicle during laparoscopic nephrectomy and nephroureterectomy using a stapler is safe. None of our patients developed an AVF during follow up.
Assuntos
Fístula Arteriovenosa/etiologia , Laparoscopia/efeitos adversos , Nefrectomia/efeitos adversos , Complicações Pós-Operatórias/etiologia , Grampeamento Cirúrgico/efeitos adversos , Ureter/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fístula Arteriovenosa/diagnóstico por imagem , Angiografia por Tomografia Computadorizada , Humanos , Ligadura , Pessoa de Meia-Idade , Nefrectomia/métodos , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
Reprogramming of immunosuppressive tumor microenvironment (TME) by targeting alternatively activated tumor associated macrophages (M2TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Tregs), represents a promising strategy for developing novel cancer immunotherapy. Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite and mediator of chronic inflammation, has emerged as a powerful immunosuppressor in the TME through engagement with one or more of its 4 receptors (EP1-EP4). We have developed E7046, an orally bioavailable EP4-specific antagonist and show here that E7046 has specific and potent inhibitory activity on PGE2-mediated pro-tumor myeloid cell differentiation and activation. E7046 treatment reduced the growth or even rejected established tumors in vivo in a manner dependent on both myeloid and CD8+ T cells. Furthermore, co-administration of E7046 and E7777, an IL-2-diphtheria toxin fusion protein that preferentially kills Tregs, synergistically disrupted the myeloid and Treg immunosuppressive networks, resulting in effective and durable anti-tumor immune responses in mouse tumor models. In the TME, E7046 and E7777 markedly increased ratios of CD8+granzymeB+ cytotoxic T cells (CTLs)/live Tregs and of M1-like/M2TAM, and converted a chronic inflammation phenotype into acute inflammation, shown by substantial induction of STAT1/IRF-1 and IFNγ-controlled genes. Notably, E7046 also showed synergistic anti-tumor activity when combined with anti-CTLA-4 antibodies, which have been reported to diminish intratumoral Tregs. Our studies thus reveal a specific myeloid cell differentiation-modifying activity by EP4 blockade and a novel combination of E7046 and E7777 as a means to synergistically mitigate both myeloid and Treg-derived immunosuppression for cancer treatment in preclinical models.
RESUMO
BACKGROUND: The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development. METHODS: Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by ß-galactosidase activity staining. RESULTS: Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated ß-galactosidase activity. CONCLUSIONS: Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.
Assuntos
Antineoplásicos/uso terapêutico , Carcinogênese/genética , Proliferação de Células/genética , Ciclina A1/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/genética , Paclitaxel/uso terapêutico , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Senescência Celular , Doxorrubicina/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Ovarianas/tratamento farmacológico , Fosfoproteínas/genética , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Transglutaminases/genéticaRESUMO
Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A-treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a (3)H-labeled apratoxin A probe and specific Sec 61α/ß antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. Mol Cancer Ther; 15(6); 1208-16. ©2016 AACR.
Assuntos
Antineoplásicos/toxicidade , Depsipeptídeos/toxicidade , Neoplasias/tratamento farmacológico , Pâncreas/efeitos dos fármacos , Canais de Translocação SEC/metabolismo , Células A549 , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/farmacocinética , Humanos , Células MCF-7 , Dose Máxima Tolerável , Camundongos , Transplante de Neoplasias , Neoplasias/metabolismo , Especificidade de Órgãos , Ligação Proteica , RatosRESUMO
PURPOSE: To determine whether agents that target topoisomerase I and II could be administered sequentially. DESIGN: A Phase I study was conducted to evaluate sequential treatment with bolus IV doxorubicin followed 48 h later by topotecan given as a 30-min i.v. infusion on 3 consecutive days, with additional cycles of therapy repeated every 3 weeks. Characteristics of the 22 patients entered into the study were: 13 male and 9 female; median age, 49.5 (range 33-66) years; Eastern Cooperative Oncology Group performance status, 0-1; and normal cardiac, hematological, hepatic, and renal function. All patients had received prior therapy (median >or=2 prior regimens). RESULTS: The maximum tolerated dose of the combination was 25 mg/m(2) doxorubicin and 5.25 mg/m(2) topotecan (1.75 mg/m(2)/day x 3). Neutropenia was the dose-limiting toxicity. Attempts to further escalate the dose using 5 microg/kg granulocyte colony-stimulating factor proved unsuccessful because of thrombocytopenia. Among the 17 patients who were evaluable for response, 6 had a partial response, and 4 showed evidence of disease stabilization. The partial responses occurred in patients with small cell lung cancer (3 of 7), non-small cell lung cancer (1 of 6), esophageal adenocarcinoma (1 of 2), and ovarian carcinoma (1 of 1), and it lasted for 3-6 months. Administration of doxorubicin 2 days before topotecan did not alter topotecan pharmacokinetics. Changes in topoisomerase mRNA levels were observed during chemotherapy. CONCLUSIONS: The sequential combination of doxorubicin followed by topotecan is highly active in several chemotherapy refractory long, ovary, and esophageal cancers. Despite significant neutropenia, toxicity is manageable and well tolerated. Phase II trials to further evaluate the efficacy of this promising combination regimen against non-Hodgkin's lymphoma and lung cancer have been initiated.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Idoso , Antígenos de Neoplasias , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Área Sob a Curva , Primers do DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Humanos , Infusões Intravenosas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Segurança , Topotecan/administração & dosagem , Topotecan/efeitos adversos , Topotecan/farmacocinética , Resultado do TratamentoRESUMO
Expression profiling to characterize cancer pharmacology has become a new approach to discover novel molecular targets for prognostic markers and cancer therapy. In a study to compare the global RNA expression profiles between primary and recurrent ovarian tumors from the same patient, we have identified XIST (inactive X chromosome-specific transcripts) as the most differentially expressed gene that was down-regulated in the recurrent tumor. XIST encodes a spliced noncoding polyadenylated transcript that is unique in being expressed exclusively from the inactive X chromosome and is involved in the X-inactivation process. Subsequent characterization of XIST expression in a panel of female cancer cell lines showed that the expression level of XIST correlates significantly with Taxol sensitivity. The clinical relevance of this observation is demonstrated by the strong association between XIST RNA levels and disease-free periods of ovarian cancer patients in a group of 21 ovarian cancer cases with Taxol in the therapeutic regiments. Cytogenetic studies on ovarian cancer cell lines indicated that loss of inactive X chromosome is one mechanism for the loss of XIST transcripts in the cell lines. Our data suggest that XIST expression may be a potential marker for chemotherapeutic responses in ovarian cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/tratamento farmacológico , RNA não Traduzido/biossíntese , Fatores de Transcrição/biossíntese , Antineoplásicos Fitogênicos/farmacologia , Coloração Cromossômica , DNA Complementar/metabolismo , Regulação para Baixo , Feminino , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Concentração Inibidora 50 , Modelos Lineares , Análise de Sequência com Séries de Oligonucleotídeos , Paclitaxel/farmacologia , Poliadenilação , RNA/metabolismo , RNA Longo não Codificante , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/ß-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/ß-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in ß-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/ß-catenin signaling addicted tumors. E7449 is currently in early clinical development.
Assuntos
Proliferação de Células/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Isoquinolinas/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Quinazolinonas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Azo , Western Blotting , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoquinolinas/química , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Neoplasias/genética , Neoplasias/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Quinazolinonas/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tanquirases/antagonistas & inibidores , Tanquirases/genética , Tanquirases/metabolismo , Temozolomida , Carga Tumoral/efeitos dos fármacos , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reduced expression of topoisomerase II is one of the mechanisms observed in cell lines and clinical samples that are resistant to topoisomerase II-targeting agents. The Chinese hamster lung cell line DC-3F/9-OH-E made resistant to 9-OH ellipticine and cross-resistant to other topoisomerase II inhibitors has previously been shown to express lower level of topoisomerase IIalpha isoform, than the parental DC-3F cell line. We have shown here that topoisomerase IIalpha promoter activity is lower in the resistant cell line. The promoter sequence responsible for the differential expression of Chinese hamster topoisomerase IIalpha gene was localized in a small promoter region, which harbors three inverted CAAT elements (ICEs) that bind transcription factor NF-Y, two GC boxes that bind Sp1 and a TATA-like element that binds unknown factors. Immunoblot analysis of cell lysates showed that the resistant line expressed reduced levels of NF-Y subunits and attenuated level of p53. Although p53 has been reported being involved in the regulation of topoisomerase II expression, it is not responsible for the reduced topoisomerase IIalpha expression in the drug resistant line. Mutational analysis of individual elements suggested that the resistant cell line has relaxed responses to ICE mutations, and the TATA-like element plays a predominant role in the regulation of topoisomerase IIalpha. Furthermore, gel mobility shift assays showed that the resistant line has a differential binding to the novel TATA-like element, which may be responsible for the down-regulation of topoisomerase IIalpha gene.
Assuntos
DNA Topoisomerases Tipo II/genética , Regiões Promotoras Genéticas/genética , Animais , Antígenos de Neoplasias , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cricetinae , Cricetulus , Proteínas de Ligação a DNA , Regulação para Baixo/genética , Resistência a Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Elipticinas/farmacologia , Regulação da Expressão Gênica , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Several 2'-fluorinated tetrahydrouridine derivatives were synthesized as inhibitors of cytidine deaminase (CDA). (4R)-2'-Deoxy-2',2'-difluoro-3,4,5,6-tetrahydrouridine (7a) showed enhanced acid stability over tetrahydrouridine (THU) 5 at its N-glycosyl bond. As a result, compound 7a showed an improved oral pharmacokinetic profile with a higher and more reproducible plasma exposure in rhesus monkeys compared to 5. Co-administration of 7a with decitabine, a CDA substrate, boosted the plasma levels of decitabine in rhesus monkeys. These results demonstrate that compound 7a can serve as an acid-stable alternative to 5 as a pharmacoenhancer of drugs subject to CDA-mediated metabolism.
Assuntos
Citidina Desaminase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Tetra-Hidrouridina/análogos & derivados , Tetra-Hidrouridina/síntese química , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Disponibilidade Biológica , Decitabina , Desenho de Fármacos , Estabilidade de Medicamentos , Inibidores Enzimáticos/farmacocinética , Potenciais Pós-Sinápticos Excitadores , Flúor , Suco Gástrico/química , Macaca mulatta , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Tetra-Hidrouridina/farmacologiaRESUMO
Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfoma não Hodgkin/tratamento farmacológico , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Piridonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo , Domínio Catalítico/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Linfoma não Hodgkin/patologia , Masculino , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Morfolinas , Mutação Puntual , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The marine natural product (+)-spongistatin 1 is an extremely potent growth inhibitory agent having activity against a wide variety of cancer cell lines, while exhibiting low cytotoxicity against quiescent human fibroblasts. Consistent with a microtubule-targeting mechanism of action, (+)-spongistatin 1 causes mitotic arrest in DU145 human prostate cancer cells. More importantly, (+)-spongistatin 1 exhibits significant in vivo antitumor activity in the LOX-IMVI human melanoma xenograft model. (+)-Spongistatin 1 is, thus, an important class of microtubule targeting anticancer agent that warrants further investigation.
Assuntos
Macrolídeos/farmacologia , Moduladores de Tubulina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Técnicas In Vitro , Macrolídeos/química , Macrolídeos/metabolismo , Camundongos , Microscopia de Fluorescência , Estereoisomerismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismoRESUMO
Selenium binding protein 1 (SELENBP1) was identified to be the most significantly down-regulated protein in ovarian cancer cells by a membrane proteome profiling analysis. SELENBP1 expression levels in 4 normal ovaries, 8 benign ovarian tumors, 12 borderline ovarian tumors and 141 invasive ovarian cancers were analyzed with immunohistochemical assay. SELENBP1 expression was reduced in 87% cases of invasive ovarian cancer (122/141) and was significantly reduced in borderline tumors and invasive cancers (p<0.001). Cox multivariate analysis within the 141 invasive cancer tissues showed that SELENBP1 expression score was a potential prognostic indicator for unfavorable prognosis of ovarian cancer (hazard ratio [HR], 2.18; 95% CI=1.22-3.90; p=0.009). Selenium can disrupt the androgen pathway, which has been implicated in modulating SELENBP1 expression. We investigated the effects of selenium and androgen on normal human ovarian surface epithelial (HOSE) cells and cancer cells. Interestingly, SELENBP1 mRNA and protein levels were reduced by androgen and elevated by selenium treatment in the normal HOSE cells, whereas reversed responses were observed in the ovarian cancer cell lines. These results suggest that changes of SELENBP1 expression in malignant ovarian cancer are an indicator of aberration of selenium/androgen pathways and may reveal prognostic information of ovarian cancer.