RESUMO
A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.
Assuntos
Transtornos Mieloproliferativos , Baço , Aminoácidos/genética , Animais , Doenças do Gato/genética , Gatos , Proliferação de Células/genética , DNA Complementar , Interleucina-3/genética , Mastócitos/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/veterinária , Nucleotídeos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores Proteína Tirosina Quinases/genética , Baço/patologiaRESUMO
Psoralidin (PSO) is a natural phenolic coumarin extracted from the seeds of Psoralea corylifolia L. Growing preclinical evidence indicates that PSO has anti-inflammatory, anti-vitiligo, anti-bacterial, and anti-viral effects. Growth arrest-specific gene 6 (GAS6) and its receptor, Axl, modulate cellular oxidative stress, apoptosis, survival, proliferation, migration, and mitogenesis. Notably, the neuroprotective role of the GAS6/Axl axis has been identified in previous studies. We hypothesize that PSO ameliorates cerebral hypoxia/reoxygenation (HR) injury via activating the GAS6/Axl signaling. We first confirmed that PSO was not toxic to the cells and upregulated GAS6 and Axl expression after HR injury. Moreover, PSO exerted a marked neuroprotective effect against HR injury, represented by restored cell viability and cell morphology, decreased lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) generation. Furthermore, PSO pretreatment also elevated the levels of nuclear factor-related factor 2 (Nrf-2), NAD(P)H dehydrogenase quinone-1 (NQO1), heme oxygenase-1 (HO-1), silent information regulator 1 (SIRT1), peroxisome proliferator-activated receptor coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), uncoupling protein 2 (UCP2), and B-cell lymphoma 2 (BCl2) both in the condition of baseline and HR injury. However, GAS6 siRNA or Axl siRNA inhibited the neuroprotective effects of PSO. Our findings suggest that PSO pretreatment attenuated HR-induced oxidative stress, apoptosis, and mitochondrial dysfunction in neuroblastoma cells through the activation of GAS6/Axl signaling.
Assuntos
Hipóxia Encefálica , Fármacos Neuroprotetores , Benzofuranos , Cumarínicos/farmacologia , Humanos , Hipóxia , Peptídeos e Proteínas de Sinalização Intercelular , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.
Assuntos
Receptores Proteína Tirosina Quinases , Transdução de Sinais , Membrana Celular/metabolismo , Humanos , Fosforilação , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina/metabolismoRESUMO
Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.
Assuntos
Neurônios Motores/enzimologia , Atrofia Muscular Espinal/enzimologia , Junção Neuromuscular/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Modelos Animais de Doenças , Ativação Enzimática , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Junção Neuromuscular/genética , Junção Neuromuscular/patologia , Receptores Proteína Tirosina Quinases/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.
Assuntos
Células da Medula Óssea/patologia , Regulação da Expressão Gênica , Rejeição de Enxerto/genética , Transplante de Coração/efeitos adversos , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Adulto , Animais , Células da Medula Óssea/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia , Citometria de Fluxo , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso Vascular/patologia , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso , Proteínas Proto-Oncogênicas/biossíntese , RNA/genética , Receptores Proteína Tirosina Quinases/biossíntese , Transplante Homólogo , Receptor Tirosina Quinase AxlRESUMO
SUMMARY: Diabetes mellitus can lead to structural disorders in the brain. One of the most common complications of diabetes, diabetic neuropathy is associated with central nervous system disorders. Aloe vera has anti-diabetic, antioxidant, and neuroprotective effects. This study was designed to evaluate the effects of Aloe vera gel on the hippocampus changes as well as the expression of nerve growth factor and receptors TrkA and P75 in the hippocampus of streptozotocin (STZ)-induced diabetic rats. 25 male Wistar rats were randomly divided into 5 groups including: control (normal saline), diabetic (normal saline), Aloe vera gel (400 mg/kg/day; gavage), diabetic + Aloe vera gel (400 mg/kg/day; gavage) and diabetic + insulin NPH (10 IU/kg/day; subcutaneous). Experimental diabetes was induced by streptozotocin injection (60 mg/kg; intraperitoneal). All groups treated for 8 weeks. At the end of treatment course, the rat brains were removed for measuring the expression of nerve growth factor, p75 and TrkA receptors were evaluated in the hippocampus. Diabetes induction after 8 weeks caused NGF and P75 expression levels in the diabetic group than other groups significantly increased (p<0.05). The TrkA receptor expression in the diabetic group compared with the control had a significant reduction (p<0.05). On the other hand in the diabetic group receiving Aloe vera gel expression of NGF and P75 expression levels compared to the diabetic group was significantly reduced (p<0.05) and the TrkA receptor expression compared with the diabetic group had a significant increase (p<0.05). The results showed that oral administration of Aloe vera gel in diabetic rats ameliorates diabetes-induced hyperglycemia. On the other hand, Aloe vera gel cause modulation of the expression of NGF neurotrophic factor via increased expression of TrkA receptor-specific and non-specific receptor down-regulation of P75 in the hippocampus of STZ-induced diabetic rats.
RESUMEN: La diabetes mellitus puede provocar trastornos estructurales en el cerebro. Es una de las complicaciones más comunes de la diabetes y la neuropatía diabética y está relacionada con trastornos del sistema nervioso central. El Aloe vera tiene efectos antidiabéticos, antioxidantes y neuroprotectores. Este estudio fue diseñado para evaluar los efectos del gel de Aloe vera en los cambios del hipocampo, así como la expresión del factor de crecimiento nervioso y los receptores TrkA y P75 en el hipocampo de ratas diabéticas inducidas por estreptozotocina (STZ). Se dividieron al azar 25 ratas Wistar macho en 5 grupos de: control (solución salina normal), diabéticos (solución salina normal), gel de Aloe vera (400 mg / kg / día; sonda), diabéticos + gel de Aloe vera (400 mg / kg / día; sonda) y diabéticos + insulina NPH (10 UI / kg / día; subcutánea). La diabetes experimental fue inducida por inyección de estreptozotocina (60 mg / kg; intraperitoneal). Todos los grupos fueron tratados durante 8 semanas. Al final del tratamiento, se extrajeron los cerebros de las ratas para medir la expresión del factor de crecimiento nervioso y se evaluaron los receptores p75 y TrkA en el hipocampo. La inducción de diabetes después de 8 semanas provocó que los niveles de expresión de NGF y P75 en el grupo de diabéticos aumentaran significativamente en comparación con otros grupos (p <0,05). La expresión del receptor TrkA en el grupo diabético comparado con el control tuvo una reducción significativa (p <0,05). Por otro lado, el grupo de ratas diabéticas que recibieron la expresión en gel de Aloe vera de NGF y los niveles de expresión de P75 en comparación con el grupo de ratas diabéticas se redujo significativamente (p <0,05) y la expresión del receptor de TrkA en comparación con el grupo de ratas diabéticas tuvo un aumento significativo (p <0,05). Los resultados mostraron que la administración oral de gel de Aloe vera en ratas diabéticas mejora la hiperglucemia inducida por la diabetes. Por otro lado, el gel de Aloe vera causa modulación de la expresión del factor neurotrófico NGF a través del aumento de la expresión de receptor TrkA específico y no específico y regulación negativa del receptor de P75 en el hipocampo de ratas diabéticas inducidas por STZ.
Assuntos
Animais , Masculino , Ratos , Extratos Vegetais/administração & dosagem , Fator de Crescimento Neural/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Aloe/química , Hipocampo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Administração Oral , Ratos Wistar , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1ß, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.
Assuntos
Macrófagos/metabolismo , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Miocardite/etiologia , Miocardite/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Inflamassomos/metabolismo , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptor Cross-Talk , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , c-Mer Tirosina Quinase/deficiência , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, although its cytotoxicity might be hampered by autophagy. In this study, we examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells. METHODS: The effects of PON in inducing autophagy were determined both in vitro, using SK-N-BE(2), SH-SY5Y, and IMR-32 human neuroblastoma cell lines, and in vivo, using zebrafish and mouse models. Single and combined treatments with chloroquine (CQ)-a blocking agent of lysosomal metabolism and autophagic flux-and PON were conducted, and the effects on cell viability were determined using metabolic and immunohistochemical assays. The activation of the autophagic flux was analyzed through immunoblot and protein arrays, immunofluorescence, and transmission electron microscopy. Combination therapy with PON and CQ was tested in a clinically relevant neuroblastoma mouse model. RESULTS: Our results confirm that, in neuroblastoma cells and wild-type zebrafish embryos, PON induces the accumulation of autophagy vesicles-a sign of autophagy activation. Inhibition of autophagic flux by CQ restores the cytotoxic potential of PON, thus attributing to autophagy a cytoprotective nature. In mice, the use of CQ as adjuvant therapy significantly improves the anti-tumor effects obtained by PON, leading to ulterior reduction of tumor masses. CONCLUSIONS: Together, these findings support the importance of autophagy monitoring in the treatment protocols that foresee PON administration, as this may predict drug resistance acquisition. The findings also establish the potential for combined use of CQ and PON, paving the way for their consideration in upcoming treatment protocols against neuroblastoma.
Assuntos
Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Neuroblastoma/tratamento farmacológico , Piridazinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Camundongos , Neuroblastoma/genética , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Axl, a member of the TAM (Tyro3, AXL, Mer) receptor tyrosine kinase family, plays critical roles in cell growth, proliferation, apoptosis, and migration. In the present study, we demonstrated that the anti-cancer activity of bufalin, a major bioactive component of the Chinese traditional medicine Chan Su, is mediated by the down-regulation of Axl in non-small-cell lung cancer (NSCLC) cells. We observed the inhibitory effect of bufalin on the proliferation of A549 and H460 NSCLC cells and the clonogenicity of these cells was reduced by bufalin treatment in a dose-dependent manner. Next, we found that the protein level of Axl was decreased in proportion to the concentration of bufalin in both A549 and H460 cells. Moreover, the promoter activity of the Axl gene was decreased by bufalin in a dose- and time-dependent manner, indicating that bufalin down-regulates Axl gene expression at the transcriptional level. We further examined if the anti-proliferative property of bufalin is influenced by Axl at the protein level. Axl overexpression attenuated the effect of bufalin in inhibiting cell proliferation and colony formation and inducing apoptosis in H460 cells, while knockdown of Axl gene expression induced the opposite effect. Taken together, our data indicate that the anti-proliferative and pro-apoptotic effects of bufalin were associated with the protein level of Axl, suggesting that Axl is a potent therapeutic target of bufalin in suppressing proliferation and inducing apoptosis in NSCLC cells.
Assuntos
Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Bufanolídeos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Receptor Tirosina Quinase AxlRESUMO
TRK fusions are oncogenic drivers of various adult and paediatric cancers. The first-generation TRK inhibitors, larotrectinib and entrectinib, were granted landmark, tumour-agnostic regulatory approvals for the treatment of these cancers in 2018 and 2019, respectively. Brisk and durable responses are achieved with these drugs in patients, including those with locally advanced or metastatic disease. In addition, intracranial activity has been observed with both agents in TRK fusion-positive solid tumours with brain metastases and primary brain tumours. While resistance to first-generation TRK inhibition can eventually occur, next-generation agents such as selitrectinib (BAY 2731954, LOXO-195) and repotrectinib were designed to address on-target resistance, which is mediated by emergent kinase domain mutations, such as those that result in substitutions at solvent front or gatekeeper residues. These next-generation drugs are currently available in the clinic and proof-of-concept responses have been reported. This underscores the utility of sequential TRK inhibitor use in select patients, a paradigm that parallels the use of targeted therapies in other oncogenic driver-positive cancers, such as ALK fusion-positive lung cancers. While TRK inhibitors have a favourable overall safety profile, select on-target adverse events, including weight gain, dizziness/ataxia and paraesthesias, are occasionally observed and should be monitored in the clinic. These side-effects are likely consequences of the inhibition of the TRK pathway that is involved in the development and maintenance of the nervous system.
Assuntos
Neoplasias/tratamento farmacológico , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Criança , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indazóis/farmacologia , Indazóis/uso terapêutico , Neoplasias/enzimologia , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
The fruits of Lycium barbarum are considered medicinal foods with high nutritional value and bioactivity. In this study, we aimed to evaluate the effect of a crude L. barbarum polysaccharide (LBP) and two derived fractions, LBP-1 and LBP-2, on the lifespan of Drosophila melanogaster (fruit fly). The average lifespan of fruit flies was extended by supplementing their diet with either of the three LBP preparations. In vivo analysis of antioxidant activities detected increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased malondialdehyde (MDA) levels. Dietary LBP supplements significantly reduced the mortality rate of fruit flies induced by paraquat and hydrogen peroxide. Importantly, the strongest anti-aging activity was exhibited by the LBP-2 fraction, containing arabinogalactan with a molecular weight of 9 × 104 Da. Further studies showed that the anti-aging activity of LBP was, at least in part, mediated by an age-related signaling pathway (MAPK, TOR, S6K) and the expression of longevity genes (Hep, MTH, and Rpn11).
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Longevidade/efeitos dos fármacos , Animais , Antioxidantes/análise , Catalase/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Frutas/química , Regulação da Expressão Gênica , Peróxido de Hidrogênio/toxicidade , Malondialdeído/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Peso Molecular , Paraquat/toxicidade , Extratos Vegetais/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has been a major obstacle in the treatment of non-small cell lung cancer (NSCLC) patients. AXL has been reported to mediate EGFR-TKIs. Recently, third generation EGFR-TKI osimertinib has been approved and yet its acquired resistance mechanism is not clearly understood. We found that AXL is involved in both gefitinib and osimertinib resistance using in vitro and in vivo model. In addition, AXL overexpression was correlated with extended protein degradation rate. We demonstrate targeting AXL degradation is an alternative route to restore EGFR-TKIs sensitivity. We confirmed that the combination effect of YD, an AXL degrader, and EGFR-TKIs can delay or overcome EGFR-TKIs-driven resistance in EGFR-mutant NSCLC cells, xenograft tumors, and patient-derived xenograft (PDX) models. Therefore, combination of EGFR-TKI and AXL degrader is a potentially effective treatment strategy for overcoming and delaying acquired resistance in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Terpenos/farmacologia , Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Medicamentos de Ervas Chinesas/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Aberrant activation of Axl is implicated in the progression of hepatocellular carcinoma (HCC). We explored the biologic significance and preclinical efficacy of Axl inhibition as a therapeutic strategy in sorafenib-naive and resistant HCC. METHODS: We evaluated Axl expression in sorafenib-naive and resistant (SR) clones of epithelial (HuH7) and mesenchymal origin (SKHep-1) using antibody arrays and confirmed tissue expression. We tested the effect of Axl inhibition with RNA-interference and pharmacologically with R428 on a number of phenotypic assays. RESULTS: Axl mRNA overexpression in cell lines (n = 28) and RNA-seq tissue datasets (n = 373) correlated with epithelial-to-mesenchymal transition (EMT). Axl was overexpressed in HCC compared to cirrhosis and normal liver. We confirmed sorafenib resistance to be associated with EMT and enhanced motility in both HuH7-SR and SKHep-1-SR cells documenting a 4-fold increase in Axl phosphorylation as an adaptive feature of chronic sorafenib treatment in SKHep-1-SR cells. Axl inhibition reduced motility and enhanced sensitivity to sorafenib in SKHep-1SR cells. In patients treated with sorafenib (n = 40), circulating Axl levels correlated with shorter survival. CONCLUSIONS: Suppression of Axl-dependent signalling influences the transformed phenotype in HCC cells and contributes to adaptive resistance to sorafenib, providing a pre-clinical rationale for the development of Axl inhibitors as a measure to overcome sorafenib resistance.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Sorafenibe/uso terapêutico , Adulto , Idoso , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sorafenibe/farmacologia , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Improvement in genetic characterization of Colon Cancer (CC) patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are receptor tyrosine kinases (RTKs) whose role in CC need to be better investigated. METHODS: We have analysed 37 CC patients using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP). We have confirmed the somatic nature of RET variants through Sanger sequencing and assessed RET activation status and protein expression by immunofluorescence and western-blot analyses. We have used RET mutant expression vectors to evaluate the effect of selected mutations in HEK293 cells by performing proliferation, migration and clonogenic assays. RESULTS: Among the 409 cancer-related genes included in the CCP we have focused on the RTKs. Overall, we have observed 101 different potentially damaging variants distributed across 31 RTK genes in 28 patients. The most frequently mutated RTKs were FLT4, ROS1, EPH7, ERBB2, EGFR, RET, FGFR3 and FGFR4. In particular, we have identified 4 different somatic variants in 10% of CC patients in RET proto-oncogene. Among them, we have demonstrated that the G533C variant was able to activate RET by promoting dimer formation and enhancing Y1062 phosphorylation. Moreover, we have demonstrated that RET G533C variant was able to stimulate anchorage-dependent proliferation, migration and clonogenic cell survival. Notably, the effects induced by the RET G533C variant were abolished by vandetanib. CONCLUSIONS: The discovery of pathogenic variants across RTK genes in 75% of the CC patients under analysis, suggests a previously underestimated role for RTKs in CC development. The identification of a gain-of-function RET mutation in CC highlights the potential use of RET in targeted therapy.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Idoso , Idoso de 80 Anos ou mais , Mutação com Ganho de Função , Humanos , Pessoa de Meia-Idade , Proto-Oncogene Mas , TransfecçãoRESUMO
Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Infecções por Salmonella/enzimologia , Salmonella typhimurium/efeitos dos fármacos , Tuberculose/enzimologia , Linhagem Celular , Biologia Computacional , Farmacorresistência Bacteriana , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
Anaplastic lymphoma kinase is a tyrosine kinase receptor protein belonging to insulin receptor superfamily. Gene fusions in anaplastic lymphoma kinase are associated with non-small cell lung cancer development. Hence, they are of immense importance in targeted therapies. Thus, for the treatment of non-small cell lung cancer, effective anaplastic lymphoma kinase inhibitors are of great significance. Therefore, our objective is to find hit compounds that could have better inhibitory activity than the existing anaplastic lymphoma kinase inhibitors. Keeping this in mind, in the present study pharmacophore based virtual screening was performed to identify possible anaplastic lymphoma kinase inhibitors. Initially, a five-point common pharmacophore hypothesis was generated based on twelve anaplastic lymphoma kinase inhibitors using PHASE module of Schrödinger. Subsequently, common pharmacophore hypothesis-based screening was conducted against in-trials subset of ZINC database and a total of 1000 hits were identified. The molecules obtained were further screened by three stages of docking using GLIDE software. The docking results reveal that six hit molecules showed higher glide score in comparison with the reference molecules. Finally, pharmacokinetic properties of the hit molecules were also analysed using QikProp programme. The results indicate that molecules namely videx, dexecadotril, chloramphenicol, naficillin were found to have good pharmacokinetic properties and human oral absorption. Moreover, videx, naficillin and chloramphenicol were found to have significant inhibitory activity for mutant (F1174L) anaplastic lymphoma kinase. It was also found that videx exhibited crucial interactions with the Met1199 residue of the native and mutant anaplastic lymphoma kinase protein. Furthermore, PASS algorithm predicted anti-neoplastic activity for all the four molecules. Thus these hits are found to be promising leads for anaplastic lymphoma kinase inhibitors. We believe that this study will be useful for the discovery and designing of more potent anaplastic lymphoma kinase inhibitors in the near future.
Assuntos
Inibidores de Proteínas Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Algoritmos , Quinase do Linfoma Anaplásico , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Domínio Catalítico , Cloranfenicol/química , Cloranfenicol/metabolismo , Bases de Dados de Compostos Químicos , Bases de Dados de Proteínas , Didanosina/química , Didanosina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Conformação Molecular , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Termodinâmica , Tiorfano/análogos & derivados , Tiorfano/química , Tiorfano/metabolismoRESUMO
Neurotrophic tyrosine kinase genes encode for the Trk-family proteins TrkA, TrkB, and TrkC, which have an important role in the development of the nervous system; however, they have been identified as oncogenic fusions in solid tumors (NTK-1, NTRK-2, and NTRK-3) and are associated with poor survival in lung cancer. These three new fusions can be detected by fluorescent in situ hybridization or next-generation sequencing in less than 5% of the lung tumors. There are several ongoing clinical trials of NTRK oncogenes in lung cancer and other tumors. The agents entrectinib (RXDX-101), a multi-kinase small molecule inhibitor that selectively inhibits NTRK1, NTRK2, and NTRK3, ROS1 and ALK, and LOXO-101, an ATP-competitive pan-NTRK inhibitor, have shown responses in patients with lung cancer with an acceptable toxicity profile. Although these oncogenic fusions are not very prevalent, the high prevalence of lung cancer makes these findings very relevant and suggests the feasibility of these oncogenes as targets in lung cancer. New data from Ozono and collaborators presented in this issue suggest that BDNF/TrkB signal promotes proliferating migratory and invasive phenotypes and cellular plasticity in squamous cell carcinoma (SCC) of the lung but that it also represents a druggable target that may bring hope to squamous lung cancer patients.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias de Células Escamosas/tratamento farmacológico , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Animais , Antineoplásicos/efeitos adversos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Terapia de Alvo Molecular/efeitos adversos , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptor trkA/antagonistas & inibidores , Receptor trkA/genética , Receptor trkA/metabolismo , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/antagonistas & inibidores , Receptor trkC/genética , Receptor trkC/metabolismoRESUMO
BACKGROUND: Mistletoe extract of Visucm album extract (VAE) contains many biologically active components and has been reported to be not only a complementary and alternative medicine, but also a potent therapeutic agent for many types of cancer. PURPOSE: In this study, we examined the effect of VAE on expression and activation of Axl and scrutinized the involvement of Axl in the anti-cancer activity of VAE in parental and chemo-resistant non-small cell lung cancer (NSCLC) cells. METHODS: The levels of Axl protein and mRNA were determined by Western blot analysis and RT-PCR, respectively. Phosphorylation of Axl upon Gas6 stimulation was observed by Western blot analysis. For ectopic expression or gene silencing of Axl, the recombinant plasmid, pcDNA3-Axl, or specific siRNA targeting Axl were transfected into A549 and H460 cells using Lipofectamine 2000, respectively. The anti-cancer activity of mistletoe extract was examined against the parental cells and each of their cisplatin- or erlotinib-resistant cells using trypan blue exclusion assays and colony formation assay. RESULTS: The levels of Axl mRNA were also reduced by VAE treatment, implying the transcriptional downregulation of Axl expression by VAE. In addition, the phosphorylation of Axl protein upon its ligand, Gas6, stimulation was found to be abrogated by VAE. We next found cytotoxic effect of VAE on both the parental NSCLC cells and their variants which are resistant to cisplatin (A549/CisR and H460/CisR) or erlotinib (H460/ER and H1975/ER). Treatment of these cells with VAE caused a dose-dependent decrease of cell viability and clonogenicity. This anti-proliferative effect of VAE was attenuated in Axl-overexpressing cells, while it was augmented in cells transfected Axl specific siRNA. Next, we also found that in cisplatin-resistant cells and erlotinib-resistant cells, VAE treatment decreased Axl protein level, colonogenicity. The levels of several cell cycle regulator, p21 and apoptosis related protein, X-linked inhibitor of apoptosis, was found to be induced and reduced by VAE treatment, respectively. CONCLUSION: Taken together, our data provide that VAE targets Axl to suppress cell proliferation and to circumvent cisplatin- and erlotinib-resistance in NSCLC cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Viscum album/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cloridrato de Erlotinib/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptor Tirosina Quinase AxlRESUMO
The discovery of ALK rearrangement in non-small-cell lung cancer (NSCLC) triggered rapid clinical development of a family of specific drugs targeting this alteration, called ALK inhibitors. Despite high rate of responses, the vast majority of patients treated with first-generation ALK inhibitor crizotinib will ultimately develop disease progression. The second-generation ALK inhibitor, ceritinib, is an oral, small-molecule that inhibits the ALK kinase activity with a potency 20-fold greater than crizotinib, being able to tackle some of the principal mechanisms of resistance to crizotinib. Evidences from five large prospective clinical trials have so far showed impressive activity of ceritinib in ALK inhibitor pretreated and naive NSCLC patients. This review will focus on the preclinical and clinical data available regarding ceritinib pharmacology, clinical efficacy and safety profile.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonas/uso terapêutico , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Terapia de Alvo Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Sulfonas/farmacologiaRESUMO
Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to 2nd-generation ALK inhibitors. Lorlatinib (PF-06463922) (6) is a 3rd-generation macrocyclic ALK-TKI that demonstrates many advantages over 2nd-generation ALK inhibitors. Lorlatinib has demonstrated decent kinase selectivity, promising pharmacokinetic profile, selective brain-penetration and strong antiproliferative activity in several ALK/ROS1-driven tumor models. The current review describes the activity spectrum, key events from discovery to clinical applications and the evidences that lorlatinib acts as an ALK/ROS1 inhibitor in clinical settings.