RESUMO
Tetrazanbigen (TNBG) is a novel sterol isoquinoline derivative with poor water solubility and moderate inhibitory effects on human cancer cell lines via lipoapoptosis induction. Herein, we developed a series of novel TNBG analogues with improved water solubility and antiproliferative activities. The CCK-8 assay enabled us to identify a novel compound, 14g, which strongly inhibited HepG2 and A549 cell growth with IC50 values of 0.54 and 0.47 µM, respectively. The anticancer effects might be explained by the partial activation and upregulation of PPARγ expression, as indicated by the transactivation assay and western blotting evaluation. Furthermore, the in vitro antiproliferative activity was verified in an in vivo xenograft model in which 14g strongly reduced tumor growth at a dose of 10 mg/kg. In line with these positive observations, 14g exhibited an excellent water solubility of 31.4 mg/mL, which was more than 1000-fold higher than that of TNBG (4 µg/mL). Together, these results suggest that 14g is a promising anticancer therapeutic that deserves further investigation.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos Azo/química , Compostos Azo/farmacologia , Gonanos/química , Gonanos/farmacologia , PPAR gama/agonistas , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Solubilidade , Relação Estrutura-Atividade , Ensaio Tumoral de Célula-Tronco , Vacúolos/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Type I (IFNα/ß) interferon signaling represents a critical transduction pathway involved in recognition and destruction of nascent tumor cells. Downregulation of this pathway to promote a more immunosuppressed microenvironment contributes to the ability of tumor cells to evade the immune system, a known Hallmark of Cancer. The present study investigates the progesterone receptor (PR), which is expressed in the vast majority of breast cancers, and its ability to inhibit efficient interferon signaling in tumor cells. We have shown that PR can block the interferon signaling cascade by promoting ubiquitination and degradation of STAT2. Targeting STAT2 is critical, as we show that it is an essential protein in inducing transcription of interferon-stimulated genes (ISG); shRNA-mediated knockdown of STAT2 severely abrogates the interferon response in vitro. Importantly, we were able to reverse this inhibition by treating with onapristone, an anti-progestin currently being investigated in breast cancer clinical trials. Additionally, we have found that an interferon-related gene signature (composed of ISGs) is inversely correlated with PR expression in human tumors. We speculate that PR inhibition of interferon signaling may contribute to creating an immunosuppressed microenvironment and reversal of this through anti-progestins may present a novel therapeutic target to promote immune activity within the tumor.
Assuntos
Neoplasias da Mama , Interferons , Receptores de Progesterona , Fator de Transcrição STAT2 , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Feminino , Gonanos/farmacologia , Humanos , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente TumoralRESUMO
BACKGROUND: Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ. METHODS: Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ. RESULTS: Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis. CONCLUSIONS: The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.
Assuntos
Autofagia/efeitos dos fármacos , Glioblastoma/metabolismo , Gonanos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Temozolomida/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood-brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.
Assuntos
Azóis/administração & dosagem , Doença de Depósito de Glicogênio Tipo II/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Compostos Organosselênicos/administração & dosagem , alfa-Glucosidases/metabolismo , Animais , Azóis/farmacologia , Barreira Hematoencefálica , Encéfalo/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/metabolismo , Gonanos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Injeções Intraperitoneais , Isoindóis , Masculino , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Wortmanina/farmacologia , alfa-Glucosidases/genéticaRESUMO
OBJECTIVES: TNBG-5602 is a newly synthesized compound with an isoquinoline structure. In the present study, we demonstrated the anticancer effect of TNBG-5602 in in-vitro and in-vivo models and investigated its possible anticancer mechanism. METHODS: The antiproliferation effect of TNBG-5602 in vitro was evaluated in human liver cancer cell line QGY-7701. The acute toxicity of TNBG-5602 was evaluated in mice. The anticancer activity of TNBG-5602 in vivo was assessed in a xenograft model of human liver cancer cell line QGY-7701. KEY FINDINGS: The results of CCK-8 assay showed that TNBG-5602 can effectively inhibit the proliferation of liver cancer cells in vitro. The acute toxicity test in mice showed that the LD50 of TNBG-5602 was 172 mg/kg. In a xenograft liver cancer model, TNBG-5602 could remarkably inhibit the growth of tumours. During in-vitro and in-vivo studies, we noted that TNBG-5602 could induce lipid accumulation in cancer cells and tissues. Further study indicated that the anticancer effect of TNBG-5602 may be exerted through activating peroxisome proliferator-activated receptor γ (PPARγ) and downregulating proliferating cell nuclear antigen (PCNA). CONCLUSIONS: Our results suggested that TNBG-5602 might exert potent anticancer activity through increasing the expression of PPARγ.
Assuntos
Antineoplásicos/farmacologia , Compostos Azo/farmacologia , Proliferação de Células/efeitos dos fármacos , Gonanos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , PPAR gama/metabolismo , Quinoxalinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
BACKGROUND: In PTEN-loss models, the phosphatidylinositol 3-kinase (PI3K)/AKT and androgen receptor signaling pathways cross-regulate by reciprocal feedback whereby inhibition of one activates the other, creating a rationale for co-targeting. We studied the irreversible, pan-isoform inhibitor of Class I PI-3K PX-866 singly (part A) and with abiraterone acetate (AA) in patients on AA with rising prostate-specific antigen (PSA) (part B). PATIENTS AND METHODS: The primary endpoint was lack of progression at 12 weeks. Exploratory endpoints included changes in circulating tumor cells (CTC), pharmacodynamic studies on platelets (part A), and archival tumor exploration of PTEN as predictor of response (part B). RESULTS: A total of 43 and 25 patients accrued to parts A and B, respectively. In part A, 14 (33%) patients were progression-free at 12 weeks, with 2 partial objective responses and 1 confirmed PSA response. Favorable CTC conversion (< 5 CTC/7.5 mL) occurred in 6 (24%) of 25 evaluable patients. In part B, 11 of 25 patients had measurable disease. Six (24%) patients were progression-free at 12 weeks. No objective or PSA responses were observed. For all 68 patients, the most common toxicities were diarrhea (53 patients), nausea (36), anorexia (24), fatigue (22), and vomiting (20). Among 17 patients for whom PTEN testing was possible, 3 had PTEN homozygous deletion and 14 had no change. No correlation between PTEN status and response was seen. CONCLUSIONS: PX-866 had modest single agent activity. Adding AA to PX-866 showed no evidence of resistance reversal. Strategies to combine PI3K inhibition with androgen receptor-targeted therapies could consider initiation earlier, combination with other agents, and/or recruiting a selected population.
Assuntos
Androstenos/administração & dosagem , Gonanos/administração & dosagem , Recidiva Local de Neoplasia/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Androstenos/efeitos adversos , Androstenos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Canadá , Progressão da Doença , Gonanos/efeitos adversos , Gonanos/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos dos fármacos , Análise de Sobrevida , Resultado do TratamentoRESUMO
PURPOSE: The phosphoinositide-3-kinase (PI3K) pathway is the frequently altered in human cancer. This has led to the development and study of novel PI3K inhibitors for targeted therapy and also to overcome resistance to radiotherapy. METHOD: The anti-tumour effects of PI3K inhibitors (PI-828, PI-103 and PX-866) in terms of cell proliferation, colony formation, induction of apoptosis, cell cycle arrest, invasion, autophagy, and pNF-κB/p65 translocation in SCC-4, SCC-9 and SCC-25 cells were studied by performing MTT, clonogenic, DAPI staining, propidium iodide staining, annexin-V binding, matrigel invasion, acridine orange staining and immuno-fluorescence assay. Western blot assay was performed to assess the alteration in the expression of various proteins. RESULT: PI-828 and PI-103 treatment exhibited dose-dependent inhibition of growth and proliferation of OSCC cells with a concomitant induction of apoptosis, altered cell cycle regulation and decreased invasiveness (p < 0.01). PX-866 induced apoptosis, cell cycle arrest, autophagy and a significant decrease in the invasiveness of oral cancer cells as compared to untreated cells (p < 0.01). These compounds significantly reduced expression of COX-2, cyclin-D1 and VEGF in the treated cells besides cytoplasmic accumulation of pNF-κB/p65 protein. In addition to PI3Kα, inactivation of downstream components, i.e. Akt and mTOR was seen. CONCLUSION: PI3K inhibitors such as PI-103, PI-828 and PX-866 may be developed as potential therapeutic agents for effective treatment of oral squamous cell carcinoma (OSCC) patients, associated with activated PI3K/Akt pathway.
Assuntos
Neoplasias Bucais/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Furanos/farmacologia , Furanos/uso terapêutico , Gonanos/farmacologia , Gonanos/uso terapêutico , Humanos , Neoplasias Bucais/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Persistence of latent HIV-1 in macrophages (MACs) and T-helper lymphocytes (THLs) remain a major therapeutic challenge. Currently available latency reversing agents (LRAs) are not very effective in vivo. Therefore, understanding of physiologic mechanisms that dictate HIV-1 latency/reactivation in reservoirs is clearly needed. Mesenchymal stromal/stem cells (MSCs) regulate the function of immune cells; however, their role in regulating virus production from latently-infected MACs & THLs is not known. We documented that exposure to MSCs or their conditioned media (MSC-CM) rapidly increased HIV-1 p24 production from the latently-infected U1 (MAC) & ACH2 (THL) cell lines. Exposure to MSCs also increased HIV-1 long terminal repeat (LTR) directed gene expression in the MAC and THL reporter lines, U937-VRX and J-Lat (9.2), respectively. MSCs exposed to CM from U1 cells (U1-CM) showed enhanced migratory ability towards latently-infected cells and retained their latency-reactivation potential. Molecular studies showed that MSC-mediated latency-reactivation was dependent upon both the phosphatidyl inositol-3-kinase (PI3K) and nuclear factor-κB (NFκB) signaling pathways. The pre-clinically tested inhibitors of PI3K (PX-866) and NFκB (CDDO-Me) suppressed MSC-mediated HIV-1 reactivation. Furthermore, coexposure to MSC-CM enhanced the latency-reactivation efficacy of the approved LRAs, vorinostat and panobinostat. Our findings on MSC-mediated latency-reactivation may provide novel strategies against persistent HIV-1 reservoirs.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/fisiologia , Células-Tronco Mesenquimais/metabolismo , Ativação Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Avaliação Pré-Clínica de Medicamentos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Gonanos/farmacologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , NF-kappa B/metabolismo , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Vorinostat/farmacologia , Vorinostat/uso terapêuticoRESUMO
BACKGROUND: Central alveolar hypoventilation syndromes (CHS) encompass neurorespiratory diseases resulting from congenital or acquired neurological disorders. Hypercapnia, acidosis, and hypoxemia resulting from CHS negatively affect physiological functions and can be lifethreatening. To date, the absence of pharmacological treatment implies that the patients must receive assisted ventilation throughout their lives. OBJECTIVE: To highlight the relevance of determining conditions in which using gonane synthetic progestins could be of potential clinical interest for the treatment of CHS. METHODS: The mechanisms by which gonanes modulate the respiratory drive were put into the context of those established for natural progesterone and other synthetic progestins. RESULTS: The clinical benefits of synthetic progestins to treat respiratory diseases are mixed with either positive outcomes or no improvement. A benefit for CHS patients has only recently been proposed. We incidentally observed restoration of CO2 chemosensitivity, the functional deficit of this disease, in two adult CHS women by desogestrel, a gonane progestin, used for contraception. This effect was not observed by another group, studying a single patient. These contradictory findings are probably due to the complex nature of the action of desogestrel on breathing and led us to carry out mechanistic studies in rodents. Our results show that desogestrel influences the respiratory command by modulating the GABAA and NMDA signaling in the respiratory network, medullary serotoninergic systems, and supramedullary areas. CONCLUSION: Gonanes show promise for improving ventilation of CHS patients, although the conditions of their use need to be better understood.
Assuntos
Gonanos/farmacologia , Gonanos/uso terapêutico , Progesterona/análogos & derivados , Apneia do Sono Tipo Central/tratamento farmacológico , Animais , Desogestrel/farmacologia , Desogestrel/uso terapêutico , Humanos , Progestinas/farmacologiaRESUMO
Although progesterone receptor (PR)-targeted therapies are modestly active in patients with uterine cancer, their underlying molecular mechanisms are not well understood. The clinical use of such therapies is limited because of the lack of biomarkers that predict response to PR agonists (progestins) or PR antagonists (onapristone). Thus, understanding the underlying molecular mechanisms of action will provide an advance in developing novel combination therapies for cancer patients. Nuclear translocation of PR has been reported to be ligand-dependent or -independent. Here, we identified that onapristone, a PR antagonist, inhibited nuclear translocation of ligand-dependent or -independent (EGF) phospho-PR (S294), whereas trametinib inhibited nuclear translocation of EGF-induced phospho-PR (S294). Using orthotopic mouse models of uterine cancer, we demonstrated that the combination of onapristone and trametinib results in superior antitumor effects in uterine cancer models compared with either monotherapy. These synergistic effects are, in part, mediated through inhibiting the nuclear translocation of EGF-induced PR phosphorylation in uterine cancer cells. Targeting MAPK-dependent PR activation with onapristone and trametinib significantly inhibited tumor growth in preclinical uterine cancer models and is worthy of further clinical investigation. Mol Cancer Ther; 17(2); 464-73. ©2017 AACR.
Assuntos
Antineoplásicos/uso terapêutico , Gonanos/uso terapêutico , Receptores de Progesterona/antagonistas & inibidores , Neoplasias Uterinas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Gonanos/farmacologia , Humanos , Camundongos , Camundongos Nus , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologiaRESUMO
Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-ß pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-ß pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression.
Assuntos
Ciclina D1/genética , Regulação para Baixo/efeitos dos fármacos , Gonanos/farmacologia , Cloridrato de Raloxifeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Telomerase/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Proteína Axina/genética , Proteína Axina/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Células MCF-7 , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Smad/metabolismo , Telomerase/metabolismoRESUMO
Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two- and three-dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti-invasive potential of PI3K inhibitors and that drugs such as PX-866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E-BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.
Assuntos
Melanoma/patologia , Modelos Biológicos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Gonanos/farmacologia , Indóis/farmacologia , Melanoma/enzimologia , Camundongos Endogâmicos NOD , Mutação/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Sulfonamidas/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
As a potential means to reduce proliferation of breast cancer cells, a multiple-pathway approach with no effect on control cells was explored. The human interactome being constructed by the Center for Cancer Systems Biology will prove indispensable to understanding composite effects of multiple pathways, but its discovered protein-protein interactions require characterization. Accordingly, we explored the effects of regulators of one protein on downstream targets of the other protein. MCF-7 estrogen receptor-positive (ER+) breast cancer cells were treated with raloxifene to upregulate the TGF-ß pathway and PX-866 to down-regulate the PI3K/Akt pathway. This resulted in highly significant downstream reduction of cell cycle proliferation in breast cancer cells with no significant proliferation reduction following similar treatment of noncancerous MCF10A breast epithelial cells. Reduced phosphorylation of p107 and substantial reduction of Rb phosphorylation were observed in response. The effects of reduced Rb and p107 phosphorylation were reflected in significant decline in E2F-1 transcriptional activity, which is dependent on pocket protein phosphorylation status. The reduced proliferation was related to decreased expression of cyclins, including E2F-1-regulated Cyclin E2, which was also in response to raloxifene and PX-866. All combinations of raloxifene and PX-866 produced significant or highly significant results for reduced MCF-7 cell proliferation, reduced Cyclin E2 transcription, and reduced Rb phosphorylation. These studies demonstrated that uncontrolled proliferation of ER+ breast cancer cells can be significantly reduced by combinational targeting of two relevant pathways. J. Cell. Biochem. 117: 1688-1696, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Neoplasias da Mama , Proliferação de Células/efeitos dos fármacos , Ciclinas/biossíntese , Gonanos/farmacologia , Cloridrato de Raloxifeno/farmacologia , Proteína do Retinoblastoma/metabolismo , Transcrição Gênica/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator de Transcrição E2F1/metabolismo , Feminino , Humanos , Células MCF-7RESUMO
Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Receptores de Progesterona/metabolismo , Transcrição Gênica , Cromonas/farmacologia , Gonanos/farmacologia , Células HeLa , Humanos , Complexo Mediador/metabolismo , Mifepristona/farmacologia , Modelos Biológicos , Morfolinas/farmacologia , Promegestona/farmacologia , Purinas/farmacologia , Proteínas Recombinantes/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
OBJECTIVES: Tetrazanbigen (TNBG) is a newly synthesized compound with an isoquinoline moiety, and its antitumour effects were evaluated in in-vitro and in-vivo models. METHODS: 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure the antiproliferative activity of TNBG on cancer cell lines. Antitumour activity of TNGBâ in vivo was also assessed in a xenograft model of human hepatocellular carcinoma QGY-7701 cell line. Cell cycle and cell apoptosis analysis was performed. KEY FINDINGS: TNBG exhibited strong antitumour efficacy against six human cancer cell lines with IC50 range of 2.13-8.01 µg/ml. The IC50 of TNBG on normal hepatic cells was 11.25 µg/ml. Lots of lipid droplets were observed in cytoplasm of human hepatocellular carcinoma QGY-7701 cells after treatment of TNBG. S phase arrest and apoptosis induction by TNBG were also found on QGY-7701 cells. Intraperitoneal injection of TNBG (1.5 mg/kg/day) resulted in significant decreases in tumour volume and tumour weight on nude mice bearing QGY-7701 cells xenografts. Results from pathological analysis in nude mice demonstrated that TNBG could induce lipid accumulation specifically in cancer tissue rather than in other normal organs, tissues and blood. CONCLUSIONS: These results suggested that TNBG might exert potent antitumour activity through inducing lipid accumulation in cancer cell.
Assuntos
Antineoplásicos/farmacologia , Compostos Azo/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Gonanos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Compostos Azo/administração & dosagem , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Gonanos/administração & dosagem , Humanos , Concentração Inibidora 50 , Lipídeos/química , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer and, thus, PI3K has been recognized as an attractive molecular target for novel anti-cancer therapies. However, the effect of PI3K inhibitors on T-cell function, a key component of antitumor immunity, has been scantly explored. The objective of this study was to investigate the effect on human T-cell activation of two PI3K inhibitors currently being tested in clinical trials: PX-866 and BKM120. Their activity against a leukemic T cell line was also assessed. For that purpose, Jurkat cells or anti-CD3/anti-CD28 stimulated human peripheral blood mononuclear cells were cultured in the presence of different concentrations of PX-866 or BKM120 and their effect on T-cell proliferation, apoptosis, expression of activation markers and cytokine secretion was analyzed by flow cytometry. In addition, Akt and Erk phosphorylation was analyzed by Western blotting. Both PX-866 and BKM120 decreased viability of Jurkat cells and blocked cell cycle progression. Regarding primary T cells, both compounds similarly inhibited expression of activation markers and cytokine secretion, although they did not induce apoptosis of stimulated T cells. Interestingly, we found differences in their ability to block T-cell proliferation and IL-2 secretion, exerting BKM120 a more potent inhibition. These disparate effects could be related to differences observed in PI3K/Akt and RAS/MEK/ERK signaling between PX-866 and BKM120 treated cells. Our results suggest that, when selecting a PI3K inhibitor for cancer therapy, immunosuppressive characteristics should be taken into account in order to minimize detrimental effects on immune function.
Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Gonanos/farmacologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Linfócitos T/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
The aim of this study was to examine whether progesterone (P(4)) and its antagonists, onapristone (ZK299) and mifepristone (RU486), affect the levels of PGRA and PGRB messenger RNA (mRNA) and protein in the cow uterus which may be important in understanding whether the final physiological effect evoked by an antagonist depends on PGR isoform bound to the antagonist. Endometrial slices on Days 6 to 10 and 17 to 20 of the estrous cycle were treated for 6 or 24 hours for mRNA and protein expression analysis, respectively, with P4, ZK299, or RU486 at a dose of 10(-4), 10(-5), or 10(-6) M. In the samples on Days 6 to 10 of the estrous cycle, PGRAB mRNA was stimulated by P(4) (10(-4) M; P < 0.01) and RU486 (10(-6); P < 0.001) and was decreased by ZK299 (10(-5); P < 0.05). In contrast, PGRB mRNA was decreased by the all P(4) (P < 0.01) and ZK299 (P < 0.001) doses and by two of the RU486 doses (10(-4) M; P < 0.01 and 10(-5) M; P < 0.01). In samples on Days 17 to 20 of the estrous cycle, PGRAB mRNA was stimulated by RU486 (10(-5) M; P < 0.001). PGRB mRNA was decreased by P(4) (10(-4) and 10(-5) M; P < 0.001), ZK299 (10(-4) and 10(-5) M; P < 0.001), and RU486 (10(-4) M; P < 0.01 and 10(-6) M; P < 0.001) and was increased by ZK299 (10(-6) M; P < 0.001) and RU486 (10(-5) M; P < 0.001). In samples on Days 6 to 10 of the estrous cycle, PGRB protein levels were decreased (P < 0.05) by all three ZK299 doses and by two of the RU486 doses (10(-4) M; P < 0.05 and 10(-5) M; P < 0.01). In contrast, in samples on Days 17 to 20, both PGRA and PGRB protein levels were decreased by ZK299 stimulation (10(-5) M; P < 0.05 and 10(-5) M; P < 0.01, respectively), whereas only PGRA protein levels were increased by RU486 (10(-5) M; P < 0.01). Both ZK299 and RU486 may exhibit both agonist and antagonist properties depending on which receptor isoform they affect. As a result, an increase or decrease in the expression of a particular PGR isoform will be observed.
Assuntos
Endométrio/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Gonanos/farmacologia , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Receptores de Progesterona/genética , Animais , Bovinos , Endométrio/metabolismo , Feminino , Progesterona/antagonistas & inibidores , Progesterona/metabolismo , Prostaglandinas/metabolismo , RNA Mensageiro/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Progesterone receptor and estrogen receptor participate in growth and differentiation of the different rat decidual regions. Steroid hormone receptor antagonists were used to study steroid regulation of decidualization. Here we describe a suppressive interaction between progesterone receptor (onapristone) and estrogen receptor (ICI182780) antagonists and their relation to a rescue phenomenon with concomitant regulation of Hand2, Bmp2 and p-ERK1/2 during the early decidualization steps. Phenotypes of decidua development produced by antagonist treatments were characterized by morphology, proliferation, differentiation, angiogenesis and expression of signaling molecules. We found that suppression of progesterone receptor activity by onapristone treatment resulted in resorption of the implantation sites with concomitant decrease in progesterone and estrogen receptors, PCNA, KI67 antigen, DESMIN, CCND3, CX43, Prl8a2, and signaling players such as transcription factor Hand2, Bmp2 mRNAs and p-ERK1/2. Moreover, FGF-2 and Vegfa increased as a consequence of onapristone treatment. Implantation sites from antagonist of estrogen receptor treated rats developed all decidual regions, but showed an anomalous blood vessel formation at the mesometrial part of the decidua. The deleterious effect of onapristone was partially counteracted by the impairment of estrogen receptor activity with rescue of expression levels of hormone steroid receptors, proliferation and differentiation markers, and the induction of a probably compensatory increase in signaling molecules Hand2, Bmp2 and ERK1/2 activation compared to oil treated controls. This novel drug interaction during decidualization could be applied to pathological endometrial cell proliferation processes to improve therapies using steroid hormone receptor targets.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Decídua/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Animais , Diferenciação Celular , Decídua/irrigação sanguínea , Implantação do Embrião , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Gonanos/farmacologia , Gravidez , Transporte Proteico , Ratos Wistar , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Progesterona/antagonistas & inibidores , Transdução de SinaisRESUMO
INTRODUCTION: The phosphotidylinositol-3 kinase (PI3K)/serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is frequently altered in head and neck squamous cell cancer (HNSCC). PX-866 is an oral, irreversible, pan-isoform inhibitor of PI3K. Preclinical models revealed synergy with docetaxel and a phase 1 trial demonstrated tolerability of this combination. This randomized phase 2 study evaluated PX-866 combined with docetaxel in patients with advanced, refractory HNSCC. METHODS: Patients with locally advanced, recurrent or metastatic HNSCC who had received at least one and no more than two prior systemic treatment regimens were randomized (1:1) to a combination of docetaxel (75mg/m(2) IV every 21days) with or without PX-866 (8mg PO daily; Arms A and B, respectively). The primary endpoint was progression free survival (PFS). Secondary endpoints included objective response rate (RR), overall survival (OS), toxicity, and correlation of biomarker analyses with efficacy outcomes. RESULTS: 85 patients were enrolled. There was a non-significant improvement in response rate in the combination arm (14% vs. 5%; P=0.13). Median PFS was 92days in Arm A and 82days in Arm B (P=0.42). There was no difference in OS between the two arms (263 vs. 195days; P=0.62). Grade 3 or higher adverse events were infrequent, but more common in the combination arm with respect to diarrhea (17% vs. 2%), nausea (7% vs. 0%), and febrile neutropenia (21% vs. 5%); grade 3 or higher anemia was more frequent in arm B (7% vs. 27%). PIK3CA mutations or PTEN loss were infrequently observed. CONCLUSION: The addition of PX-866 to docetaxel did not improve PFS, RR, or OS in patients with advanced, refractory HNSCC without molecular pre-selection.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Gonanos/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Metástase Neoplásica , Inibidores de Fosfoinositídeo-3 Quinase , Taxoides/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma de Células Escamosas/patologia , Docetaxel , Feminino , Gonanos/administração & dosagem , Gonanos/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Taxoides/administração & dosagemRESUMO
Epithelial tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) are typically prone to metastasis and drug resistance and contribute to a poor clinical outcome. The transcription factor ZEB1 is a known driver of EMT, and mediators of ZEB1 represent potential therapeutic targets for metastasis suppression. Here, we have shown that phosphatidylinositol 3-kinase-targeted (PI3K-targeted) therapy suppresses metastasis in a mouse model of Kras/Tp53-mutant lung adenocarcinoma that develops metastatic disease due to high expression of ZEB1. In lung adenocarcinoma cells from Kras/Tp53-mutant animals and human lung cancer cell lines, ZEB1 activated PI3K by derepressing miR-200 targets, including amphiregulin (AREG), betacellulin (BTC), and the transcription factor GATA6, which stimulated an EGFR/ERBB2 autocrine loop. Additionally, ZEB1-dependent derepression of the miR-200 and miR-183 target friend of GATA 2 (FOG2) enhanced GATA3-induced expression of the p110α catalytic subunit of PI3K. Knockdown of FOG2, p110α, and RHEB ameliorated invasive and metastatic propensities of tumor cells. Surprisingly, FOG2 was not required for mesenchymal differentiation, suggesting that mesenchymal differentiation and invasion are distinct and separable processes. Together, these results indicate that ZEB1 sensitizes lung adenocarcinoma cells to metastasis suppression by PI3K-targeted therapy and suggest that treatments to selectively modify the metastatic behavior of mesenchymal tumor cells are feasible and may be of clinical value.