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BACKGROUND: Synovial sarcoma (SS) is an aggressive soft tissue tumor with limited therapeutic options in advanced stage. SS18-SSX fusion oncogenes, which are the hallmarks of SS, cause epigenetic rewiring involving histone deacetylases (HDACs). Promising preclinical studies supporting HDAC targeting for SS treatment were not reflected in clinical trials with HDAC inhibitor (HDACi) monotherapies. We investigated pathways implicated in SS cell response to HDACi to identify vulnerabilities exploitable in combination treatments and improve the therapeutic efficacy of HDACi-based regimens. METHODS: Antiproliferative and proapoptotic effects of the HDACi SAHA and FK228 were examined in SS cell lines in parallel with biochemical and molecular analyses to bring out cytoprotective pathways. Treatments combining HDACi with drugs targeting HDACi-activated prosurvival pathways were tested in functional assays in vitro and in a SS orthotopic xenograft model. Molecular mechanisms underlying synergisms were investigated in SS cells through pharmacological and gene silencing approaches and validated by qRT-PCR and Western blotting. RESULTS: SS cell response to HDACi was consistently characterized by activation of a cytoprotective and auto-sustaining axis involving ERKs, EGR1, and the ß-endoglycosidase heparanase, a well recognized pleiotropic player in tumorigenesis and disease progression. HDAC inhibition was shown to upregulate heparanase by inducing expression of the positive regulator EGR1 and by hampering negative regulation by p53 through its acetylation. Interception of HDACi-induced ERK-EGR1-heparanase pathway by cell co-treatment with a MEK inhibitor (trametinib) or a heparanase inhibitor (SST0001/roneparstat) enhanced antiproliferative and pro-apoptotic effects. HDAC and heparanase inhibitors had opposite effects on histone acetylation and nuclear heparanase levels. The combination of SAHA with SST0001 prevented the upregulation of ERK-EGR1-heparanase induced by the HDACi and promoted caspase-dependent cell death. In vivo, the combined treatment with SAHA and SST0001 potentiated the antitumor efficacy against the CME-1 orthotopic SS model as compared to single agent administration. CONCLUSIONS: The present study provides preclinical rationale and mechanistic insights into drug combinatory strategies based on the use of ERK pathway and heparanase inhibitors to improve the efficacy of HDACi-based antitumor therapies in SS. The involvement of classes of agents already clinically available, or under clinical evaluation, indicates the transferability potential of the proposed approaches.
Assuntos
Glucuronidase/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Sarcoma Sinovial/tratamento farmacológico , Animais , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Regulação para CimaRESUMO
Pazopanib is approved for treatment of advanced soft tissue sarcomas, but primary and secondary drug resistance limits its clinical utility. We investigated the molecular mechanisms mediating pazopanib resistance in human synovial sarcoma (SS) models. We found reduced cell sensitivity to pazopanib associated with inefficient inhibition of the two critical signaling nodes, AKT and ERKs, despite strong inhibition of the main drug target, PDGFRα. In the CME-1 cell line, overactivation of IGF1 and Insulin receptors (IGF1R/InsR) sustained AKT activation and pazopanib resistance, which was overcome by a combination treatment with the double IGF1R/InsR inhibitor BMS754807. In the highly pazopanib resistant MoJo cell line, NRASQ61R mutation sustained constitutive ERK activation. Transfection of the NRAS mutant in the pazopanib sensitive SYO-1 cell line increased the drug IC50. MoJo cells treatment with pazopanib in combination with the MEK inhibitor trametinib restored ERK inhibition, synergistically inhibited cell growth, and induced apoptosis. The combination significantly enhanced the antitumor efficacy against MoJo orthotopic xenograft abrogating growth in 38% of mice. These findings identified two different mechanisms of intrinsic pazopanib resistance in SS cells, supporting molecular/immunohistochemical profiling of tumor specimens as a valuable approach to selecting patients who may benefit from rational drug combinations.
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Synovial sarcoma (SS) is an aggressive tumor with propensity for lung metastases which significantly impact patients' prognosis. New therapeutic approaches are needed to improve treatment outcome. Targeting the heparanase/heparan sulfate proteoglycan system by heparin derivatives which act as heparanase inhibitors/heparan sulfate mimetics is emerging as a therapeutic approach that can sensitize the tumor response to chemotherapy. We investigated the therapeutic potential of a supersulfated low molecular weight heparin (ssLMWH) in preclinical models of SS. ssLMWH showed a potent anti-heparanase activity, dose-dependently inhibited SS colony growth and cell invasion, and downregulated the activation of receptor tyrosine kinases including IGF1R and IR. The combination of ssLMWH and the IGF1R/IR inhibitor BMS754807 synergistically inhibited proliferation of cells exhibiting IGF1R hyperactivation, also abrogating cell motility and promoting apoptosis in association with PI3K/AKT pathway inhibition. The drug combination strongly enhanced the antitumor effect against the CME-1 model, as compared to single agent treatment, abrogating orthotopic tumor growth and significantly repressing spontaneous lung metastatic dissemination in treated mice. These findings provide a strong preclinical rationale for developing drug regimens combining heparanase inhibitors/HS mimetics with IGF1R antagonists for treatment of metastatic SS.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Heparina de Baixo Peso Molecular/farmacologia , Pirazóis/farmacologia , Receptores de Somatomedina/antagonistas & inibidores , Sarcoma Sinovial/tratamento farmacológico , Triazinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina de Baixo Peso Molecular/administração & dosagem , Heparina de Baixo Peso Molecular/metabolismo , Humanos , Camundongos SCID , Metástase Neoplásica , Pirazóis/administração & dosagem , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patologia , Sulfatos , Triazinas/administração & dosagemRESUMO
The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.
Assuntos
Heparina/análogos & derivados , Sarcoma/tratamento farmacológico , Animais , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Feminino , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sarcoma/patologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-Small Cell Lung Cancer (NSCLC) remains an aggressive and fatal disease with low responsiveness to chemotherapy, frequent drug resistance development and metastatic behavior. Platinum-based therapy is the standard of care for NSCLC with limited benefits. Since epigenetic alterations have been implicated in the aggressive behavior of lung cancer, the purpose of the present study was to examine the capability of the pan-histone deacetylase inhibitor SAHA and of ST3595, a novel hydroxamate-based compound, to interfere with the proliferative and invasive potential of NSCLC cells. We used two NSCLC cell lines (H460 and A549) and the cisplatin-resistant variants (H460/Pt and A549/Pt), to mimic a frequent clinical condition. The resistant models exhibited increased invasive properties as compared to parental cells, features associated with a wide modulation of the level of angiogenesis- and invasion-related factors in the cell conditioned media. The levels of urokinase-type plasminogen activator, IL-8, and macrophage migration inhibitory factor were increased in the conditioned media from both H460/Pt and A549/Pt cells. SAHA and ST3595 induced a strong inhibition of cell invasive properties, which was more marked after ST3595 exposure. Both HDAC inhibitors up-regulated the metastasis suppressor KiSS1 at the mRNA level. Forced expression of KiSS1 significantly decreased the invasive capability of drug-resistant cells. ST3595 displayed an anti-metastatic effect in tumors associated with decreased of phosphorylation of Src. Our data indicate that HDAC inhibitors are effective in NSCLC cell systems. The ability of ST3595 to counteract the invasive potential of resistant cells through mechanisms involving KiSS1 is an interesting novel finding.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pulmonares/patologia , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Kisspeptinas/efeitos dos fármacos , Camundongos , Camundongos Nus , FenótipoRESUMO
CONTEXT: Tyrosine kinase inhibitors represent a new treatment option for patients with advanced medullary thyroid cancer (MTC). However, cures have not been achieved with current available agents used in monotherapy. OBJECTIVE: Because RET has been shown to negatively regulate CD95 death receptor activation in preclinical models of RET-dependent MTC, we investigated the potential of the combination approach with the RET-targeting tyrosine kinase inhibitor sunitinib and cisplatin to enhance apoptosis activation through the extrinsic pathway. DESIGN: The effects of sunitinib and cisplatin were examined in human MTC cell lines harboring oncogenic RET mutations. Experiments were designed to determine drug effects on RET signaling, cell growth, apoptosis, autophagy, and tumor growth in mice and to investigate the mechanisms of the drug interaction. RESULTS: Sunitinib and cisplatin synergistically inhibited the growth of MZ-CRC-1 cells harboring the RET M918T activating mutation. The combination enhanced apoptosis activation through CD95-mediated, caspase-8-dependent pathway. Moreover, sunitinib induced a severe perturbation of the autophagic flux characterized by autophagosome accumulation and a remarkable lysosomal dysfunction, which was further enhanced, with lysosomal leakage induction, by cisplatin. Administration of the drug combination to mice xenografted with MZ-CRC-1 cells improved the antitumor efficacy, as compared with single-agent treatments, inducing complete responses in 30% of the treated mice, a significant increase in caspase-3 activation (P < .01 vs cisplatin, and P < .0005 vs sunitinib) and apoptosis in tumor cells. CONCLUSIONS: Addition of cisplatin to sunitinib potentiates apoptotic cell death and has promising preclinical activity in MTCs harboring the RET M918T oncogene.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Medular/tratamento farmacológico , Cisplatino/farmacologia , Indóis/farmacologia , Pirróis/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Medular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/uso terapêutico , Sinergismo Farmacológico , Humanos , Indóis/uso terapêutico , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/uso terapêutico , Sunitinibe , Neoplasias da Glândula Tireoide/patologiaRESUMO
Emerging evidence suggests that Ret oncoproteins expressed in medullary thyroid cancer (MTC) might evade the pro-apoptotic function of the dependence receptor proto-Ret by directly impacting the apoptosis machinery. Identification of the molecular determinants of the interplay between Ret signaling and apoptosis might provide a relevant contribution to the optimization of Ret-targeted therapies. Here, we describe the cross-talk between Ret-M918T oncogenic mutant responsible for type 2B multiple endocrine syndrome (MEN2B), and components of death receptor-mediated extrinsic apoptosis pathway. In the human MEN2B-type MTC cell line MZ-CRC-1 expressing Ret-M918T, Ret was found associated with Fap-1, known as inhibitor of the CD95 death receptor trafficking to the cell membrane, and with procaspase-8, the initiator pro-form caspase in the extrinsic apoptosis pathway. Cell treatment with the anti-tumor Ret kinase inhibitor RPI-1 inhibited tyrosine phosphorylation of procaspase-8, likely inducing its local activation, followed by downregulation of both Ret and Fap-1, and translocation of CD95 into lipid rafts. According to the resulting increase of CD95 cell surface expression, the CD95 agonist antibody CH11 enhanced RPI-1-induced cell growth inhibition and apoptosis. RET RNA interference downregulated Fap-1 protein in MZ-CRC-1 cells, whereas exogenous RET-M918T upregulated Fap-1 in HEK293 cells. Overall, these data indicate that the Ret oncoprotein exerts opposing controls on Fap-1 and CD95, increasing Fap-1 expression and decreasing CD95 cell surface expression. The functional interplay of the Ret mutant with the extrinsic apoptosis pathway provides a mechanism possibly contributing to MTC malignant phenotype and a rational basis for novel therapeutic strategies combining Ret inhibitors and CD95 agonists.
Assuntos
Apoptose , Carcinoma Medular/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , Proteínas Proto-Oncogênicas c-ret/fisiologia , Neoplasias da Glândula Tireoide/patologia , Receptor fas/fisiologia , Animais , Antineoplásicos/farmacologia , Carcinoma Medular/metabolismo , Carcinoma Neuroendócrino , Caspase 8/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Indóis/farmacologia , Camundongos , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Neoplasias da Glândula Tireoide/metabolismo , Transplante Heterólogo , Carga TumoralRESUMO
Curcumin, a natural polyphenol, has been described to exhibit effects on signaling pathways, leading to induction of apoptosis. In this study, we observed that curcumin inhibited Hsp90 activity causing depletion of client proteins implicated in survival pathways. Based on this observation, this study was designed to investigate the cellular effects of curcumin combination with the pan-HDAC inhibitors, vorinostat and panobinostat, which induce hyperacetylation of Hsp90, resulting in inhibition of its chaperone function. The results showed that, at subtoxic concentrations, curcumin markedly sensitized tumor cells to vorinostat- and panobinostat-induced growth inhibition and apoptosis. The sensitization was associated with persistent depletion of Hsp90 client proteins (EGFR, Raf-1, Akt, and survivin). In conclusion, our findings document a novel mechanism of action of curcumin and support the therapeutic potential of curcumin/HDAC inhibitors combination, because the synergistic interaction was observed at pharmacologically achievable concentrations, which were ineffective when each drug was used alone.
Assuntos
Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Indóis , Panobinostat , Relação Estrutura-Atividade , Células Tumorais Cultivadas , VorinostatRESUMO
ST1968 (namitecan), a novel 7-modified hydrophilic camptothecin, was found to be effective against tumor models relatively resistant to topotecan and irinotecan. Based on this observation, this study was designed to investigate the cellular and antitumor effects of ST1968 in a subline of A431, squamous cell carcinoma, selected for resistance to topotecan (A431/TPT). This model was characterized by a slow growth rate, associated with downregulation of EGFR and topoisomerase I. In contrast to other camptothecins (SN38 and gimatecan), ST1968 was able to overcome almost completely the resistance at cellular level. The cellular pharmacokinetics indicated a comparable accumulation and retention of ST1968 in sensitive and resistant cells, in spite of expression of the efflux transporter, P-glycoprotein, in resistant cells. The uptake and retention of topotecan were dramatically reduced in both tumor cell lines, but more evident in the resistant one. In contrast to topotecan, ST1968 retained an outstanding efficacy in vivo against the resistant tumor (A431/TPT). The results are consistent with the interpretation that ST1968 was able to overcome the most relevant mechanisms associated with the development of topotecan resistance (i.e., slow proliferation and target downregulation) owing to its peculiar pharmacokinetic behaviour.
Assuntos
Camptotecina/análogos & derivados , Carcinoma de Células Escamosas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Topotecan/uso terapêutico , Animais , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Humanos , Camundongos , Camundongos Nus , Topotecan/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Vacuolar-H(+)-ATPase plays a critical role in the cellular balance of protons, thus regulating intracellular pH and contributing to apoptosis resistance, drug resistance, and invasive and metastatic behavior of cells. NiK-12192, a vacuolar-H(+)-ATPase inhibitor, caused a reduction in the volume and/or acidity of lysosomes, a polarization of alphavbeta5 integrin distribution, and a number of floating live cells, whereas signs of apoptosis appeared only after 72 h of treatment. In conclusion, NiK-12192, by affecting vacuolar- H(+)-ATPase activity (and intracellular pH), causes a modification of structures crucial for cell adhesion and induces cell death, likely by a modality involving an anoikis-mediated apoptosis.
Assuntos
Benzamidas/farmacologia , Indóis/farmacologia , Lisossomos/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/ultraestrutura , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HCT116 , Células HL-60 , Células HT29 , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Concentração Inibidora 50 , Lisossomos/química , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Receptores de Vitronectina/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The identification of proliferation/survival pathways constitutively activated by genetic alterations in multiple myeloma (MM), or sustained by the bone marrow (BM) microenvironment, provides novel opportunities for the development of targeted therapies. The deregulated function of protein tyrosine kinases plays a critical role in driving MM malignant phenotype. We investigated the effects of the multi-target tyrosine kinase inhibitor RPI-1 in a panel of human MM cell lines, including t(4;14) positive cell lines expressing the TK receptor FGF-R3. Cells harboring FGF-R3 activating mutations (KMS11 and OPM2) displayed the highest sensitivity to RPI-1 antiproliferative effect. The stimulating effect of the aFGF ligand was abrogated in cells harboring a non-constitutively active receptor. Drug treatment inhibited activation and expression of the FGF-R3(Y373C) mutant as well as aFGF-dependent signaling involving AKT and ERKs. Inhibition of JAK2, an additional RPI-1 target, resulted in STAT3 inactivation. Blockade of these proliferation/survival pathways was associated with caspase-dependent apoptosis. Moreover, drug treatment abrogated proliferative and pro-invasive stimuli provided by conditioned medium from mesenchymal stromal cells. Gene expression profile of KMS11 cells showed 22 upregulated and 52 downregulated genes upon RPI-1 treatment, with an early modulation of genes implicated in MM pathobiology such as SAT-1, MYC, MIP-1alpha/beta, FGF-R3, and the growth factor receptor B-cell maturation antigen (BCMA). Thus, concomitant blockade of FGF-R3 and JAK2 results in inhibition of several MM-promoting pathways, including BCMA-regulated signaling, and downregulation of disease-associated proteins. These data may have therapeutic implications in the design of treatment strategies resulting in the concomitant inhibition of FGF-R3 and JAK2 signaling pathways in t(4;14) MM.
Assuntos
Antígeno de Maturação de Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Janus Quinase 2/metabolismo , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Apoptose , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismoRESUMO
The cell ability of tumor cells to tolerate stress conditions is a typical feature of solid tumors. In particular, the resistance to oxidative stress of melanoma cells likely contributes to their intrinsic drug resistance. In an attempt to develop novel strategies for overcoming the mechanisms of cellular protection against oxidative stress, in this study we have explored the efficacy of the combination of two prooxidant agents in two human melanoma cell clones. The selected clones are characterized by a marked difference in expression of gamma-glutamyltransferase, which is known to produce a persistent low level of oxidative stress resulting in the stimulation of protective systems. The gamma-glutamyltransferase-overexpressing clone exhibited a low susceptibility to arsenic trioxide-induced apoptosis, associated with low reactive oxygen species induction and increased catalase activity. The combination of arsenic trioxide with subtoxic concentrations of ascorbic acid resulted in a sensitization to apoptotic cell death. The expression of protective mechanisms, in particular catalase activity, accounted for the behavior of the resistant clone. The sensitization achieved by the combination was associated with a cellular response involving the ASK1/p38 axis, which is implicated in the regulation of catalase expression and the activation of apoptotic signals. In conclusion, the results of our study provide evidence that a rational combination of prooxidant agents may be effective in overcoming cellular tolerance to oxidative stress.
Assuntos
Arsenicais/uso terapêutico , Ácido Ascórbico/metabolismo , Inibidores do Crescimento/uso terapêutico , Melanoma/tratamento farmacológico , Óxidos/uso terapêutico , gama-Glutamiltransferase/metabolismo , Apoptose , Trióxido de Arsênio , Catalase/metabolismo , Linhagem Celular Tumoral , Citoproteção , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Estresse Oxidativo , gama-Glutamiltransferase/genéticaRESUMO
Activation of the RET gene by chromosomal rearrangements generating RET/PTC oncogenes is a frequent, early, and causative event in papillary thyroid carcinoma (PTC). We have previously shown that, in human primary thyrocytes, RET/PTC1 induces a transcriptional program including the MET proto-oncogene. In PTCs, beta-catenin is frequently mislocated to the cytoplasm nucleus. We investigated the interplay between Ret/ptc1 signaling and Met in regulating the proinvasive phenotype and beta-catenin localization in cellular models of human PTC. Here, we show that Met protein is expressed and is constitutively active in human thyrocytes exogenously expressing RET/PTC1 as well as a mutant (Y451F) devoid of the main Ret/ptc1 multidocking site. Both in transformed thyrocytes and in the human PTC cell line TPC-1, Ret/ptc1-Y451-dependent signaling and Met cooperated to promote a proinvasive phenotype. Accordingly, gene/functional silencing of either RET/PTC1 or MET abrogated early branching morphogenesis in TPC-1 cells. The same effect was obtained by blocking the common downstream effector Akt. Y451 of Ret/ptc1 was required to promote proliferation and nuclear translocation of beta-catenin, suggesting that these oncogene-driven effects are Met-independent. Pharmacologic inhibition of Ret/ptc1 and Met tyrosine kinases by the multitarget small molecule RPI-1 blocked cell proliferation and invasive ability and dislocated beta-catenin from the nucleus. Altogether, these results support that Ret/ptc1 cross talks with Met at transcriptional and signaling levels and promotes beta-catenin transcriptional activity to drive thyrocyte neoplastic transformation. Such molecular network, promoting disease initiation and acquisition of a proinvasive phenotype, highlights new options to design multitarget therapeutic strategies for PTCs.
Assuntos
Núcleo Celular/metabolismo , Transformação Celular Neoplásica/genética , Proteínas Proto-Oncogênicas c-ret/fisiologia , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento/genética , Glândula Tireoide/patologia , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Adesão Celular/genética , Proliferação de Células , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Células NIH 3T3 , Invasividade Neoplásica , Fenótipo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-met , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento/fisiologia , Glândula Tireoide/metabolismo , Regulação para Cima/fisiologia , beta Catenina/fisiologiaRESUMO
The extracellular gamma-glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. This study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H(2)O(2). Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Melanoma/enzimologia , Estresse Oxidativo/fisiologia , gama-Glutamiltransferase/metabolismo , Apoptose/fisiologia , Western Blotting , Catalase/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
On the basis of the evidence that vacuolar H(+)-ATPase is implicated in the development of the metastatic phenotype, we have explored the possibility to target the enzyme function in an attempt to control the metastatic behavior of tumor cells. In this study, we used an indole derivative, NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], recently identified as an effective inhibitor of vacuolar H(+)-ATPase, as a potential antimetastatic agent in the treatment of NSCLC H460 xenograft, which is able to induce lung metastases in mice. Oral administration of NiK-12192 caused a significant inhibition of formation of spontaneous metastases. In contrast, the drug exhibited a negligible effect on the development of artificial metastases (i.e., after i.v. injection of tumor cells), thus supporting that the drug affects the early events of the metastatic process (e.g., migration and invasion). Cellular effects are consistent with this interpretation. In conclusion, the available results show for the first time that a vacuolar H(+)-ATPase inhibitor is effective in modulation of the metastatic behavior of a lung carcinoma, supporting its potential therapeutic interest as a novel treatment approach.
Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Indóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Indóis/farmacologia , Integrina alfaVbeta3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Topotecan/uso terapêutico , ATPases Vacuolares Próton-Translocadoras/metabolismo , Cicatrização/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The proto-oncogene c-myc is involved in multiple cell pathways with opposite effects on cell outcome of death or proliferation. It has been proposed that these different roles depend on the sequestration of c-Myc protein in cellular compartments and/or its phosphorylation. We speculated that subcellular localization of c-Myc protein and of its phosphorylated form (P-c-Myc) could have a role in the different response to paclitaxel (PTX) in two prostate carcinoma cell lines, PC3 and DU145, which undergo either multinucleation or c-myc-dependent apoptosis, respectively. c-myc is amplified only in PC3, but a similar extent of c-Myc phosphorylation was observed in both cell lines after PTX treatment. We found that PTX-induced upregulation of c-myc in DU145 cells, not occurring in PC3 cells, cannot be ascribed to a different protein localization, and that a comparable c-Myc and P-c-Myc nuclear translocation occurs in both cell lines after drug treatment. Thus, subcellular localization of c-Myc and P-c-Myc is not crucial in determining the mode of cell death in these prostate carcinoma cell lines.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma/tratamento farmacológico , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proto-Oncogene MasRESUMO
Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (-75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination.
Assuntos
Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/irrigação sanguínea , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lipophilicity of camptothecins derivatives has been reported to improve the stability of the lactone ring and to favor rapid uptake and intracellular accumulation. Recently, a novel series of lipophilic camptothecins substituted at position 7 was developed, and gimatecan (ST1481) was selected for clinical development on the basis of some favorable features, including potent cytotoxicity and the unique feature of the lack of recognition by breast cancer resistance-associated protein (BCRP). In this work the intrinsic fluorescence properties of this compound were exploited to investigate its intracellular disposition in comparison with the water-soluble camptothecin, topotecan (TPT), in HT-29 colon carcinoma cells and in a subline, HT-29/Mit, selected for resistance to mitoxantrone and overexpressing BCRP. The study was performed at single-cell level by means of microspectrofluorometry and fluorescence image analysis. The results indicated a quite different subcellular localization of TPT ST1481, since TPT localized mainly in mitochondria, whereas gimatecan exhibited a lysosomal localization. An increased persistence of DNA damage in gimatecan-treated cells was consistent with the interpretation that lysosomes represent a store of active drug. In contrast to gimatecan, which showed a similar localization in HT-29 cells and in the mitoxantrone-resistant subline, the cellular pharmacokinetic of TPT was markedly influenced by overexpression of BCRP protein in the resistant subline. In conclusion, the present results indicating a quite different behavior of the two camptothecins suggest that, apart from intracellular accumulation, subcellular distribution plays a role in their cytotoxic potency and contributes to their pharmacological features.