RESUMO
High rates of recurrence and treatment resistance in the most common malignant adult brain cancer, glioblastoma (GBM), suggest that monotherapies are not sufficiently effective. Combination therapies are increasingly pursued, but the possibility of adverse drug-drug interactions may preclude clinical implementation. Developing single molecules with multiple targets is a feasible alternative strategy to identify effective and tolerable pharmacotherapies for GBM. Here, we report the development of a novel, first-in-class, dual aurora and lim kinase inhibitor termed F114. Aurora kinases and lim kinases are involved in neoplastic cell division and cell motility, respectively. Due to the importance of these cellular functions, inhibitors of aurora kinases and lim kinases are being pursued separately as anti-cancer therapies. Using in vitro and ex vivo models of GBM, we found that F114 inhibits GBM proliferation and invasion. These results establish F114 as a promising new scaffold for dual aurora/lim kinase inhibitors that may be used in future drug development efforts for GBM, and potentially other cancers.
Assuntos
Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase B/antagonistas & inibidores , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Quinases Lim/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Quinases Lim/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
LIMK1 and LIMK2 are involved in the regulation of cellular functions that depend on the dynamics of actin cytoskeleton. Disregulation of LIM kinases has been associated with diseases, such as tumor progression and metastasis, viral infection, and ocular diseases. Motivated by this, numerous studies have been carried out to discover small organic molecules capable of inhibiting LIM kinase effectively and selectively. In this review, a comprehensive survey of small organic molecules for LIM kinase inhibitors is reported, together with SAR study results, and the synthesis of these inhibitors.
Assuntos
Quinases Lim , Humanos , Quinases Lim/antagonistas & inibidores , FosforilaçãoRESUMO
Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.
Assuntos
Quinases Lim/antagonistas & inibidores , Quinases Lim/química , Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Biologia Computacional , Simulação por Computador , Desenvolvimento de Medicamentos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Humanos , Técnicas In Vitro , Ligantes , Quinases Lim/metabolismo , Aprendizado de Máquina , Simulação de Acoplamento Molecular , Invasividade Neoplásica/prevenção & controle , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Farmacologia em Rede/estatística & dados numéricos , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Interface Usuário-ComputadorRESUMO
Lung cancer is the leading cause of cancer-related deaths. LIM domain kinase (LIMK) 1 is a member of serine/threonine kinase family and highly expressed in various cancers. Luteolin, a polyphenolic plant flavonoid, has been reported to suppress tumour proliferation through inducing apoptosis and autophagy via MAPK activation in glioma. However, the mechanism of luteolin on suppressing lung cancer growth is still unclear. We found that luteolin targeted LIMK1 from the in silico screening and significantly inhibited the LIMK1 kinase activity, which was confirmed with pull-down binding assay and computational docking models. Treatment with luteolin inhibited lung cancer cells anchorage-independent colony growth and induced apoptosis and cell cycle arrest at G1 phase. Luteolin also decreased the expression of cyclin D1 and increased the levels of cleaved caspase-3 by down-regulating LIMK1 signalling related targets, including p-LIMK and p-cofilin. Furthermore, luteolin suppressed the lung cancer patient-derived xenograft tumour growth by decreasing Ki-67, p-LIMK and p-cofilin expression in vivo. Taken together, these results provide insight into the mechanism that underlies the anticancer effects of luteolin on lung cancer, which involved in down-regulation of LIMK1 and its interaction with cofilin. It also provides valuable evidence for translation towards lung cancer clinical trials with luteolin.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinases Lim/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Luteolina/farmacologia , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Técnicas In Vitro , Quinases Lim/genética , Quinases Lim/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Peritoneal metastasis is a common form of metastasis among advanced gastric cancer patients. In this study, we reported the identification of LIM domain kinase 1 (LIMK1) as a promoter of gastric cancer peritoneal metastasis, and its potential to be a therapeutic target of dabrafenib (DAB). Using transcriptomic sequencing of paired gastric cancer peritoneal metastasis, primary tumors, and normal gastric tissues, we first unveiled that LIMK1 is selectively up-regulated in metastatic tumors. Increased LIMK1 in gastric cancer peritoneal metastasis was validated by immunohistochemistry analysis of an independent patient cohort. In vitro functional studies demonstrated that LIMK1 knockout or knockdown significantly inhibited cell migration and invasion of gastric cancer cells. LIMK1 knockout also abrogated peritoneal and liver metastases of gastric cancer cells in nude mice in vivo. Dabrafenib, a small molecule targeting LIMK1, was found to decrease cell migration and invasion of gastric cancer cells in vitro and abolish peritoneal and liver metastasis formation in vivo. Mechanistically, either LIMK1 knockout or Dabrafenib inhibited LIMK1 expression and phosphorylation of its downstream target cofilin. Taken together, our results demonstrated that LIMK1 functions as a metastasis promoter in gastric cancer by inhibiting LIMK1-p-cofilin and that Dabrafenib has the potential to serve as a novel treatment for gastric cancer peritoneal metastasis.
Assuntos
Quinases Lim/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Imidazóis/farmacologia , Quinases Lim/antagonistas & inibidores , Quinases Lim/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Metástase Neoplásica , Oximas/farmacologia , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Análise de Sequência de RNA , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Taxa de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with FLT3-ITD mutated (FLT3-ITD+) Acute Myeloid Leukemia (AML), have frequently relapsed or refractory disease and FLT3-ITD+ inhibitors have limited efficacy. Rho kinases (ROCK) are constitutively activated by FLT3-ITD+ in AML via PI3 kinase and Rho GTPase. Upon activation by ROCK, LIM kinases (LIMK) inactivate cofilin by phosphorylation which affects cytoskeleton dynamics, cell growth and apoptosis. LIMK inhibition leads to cofilin activation via dephosphorylation and activated cofilin localizes to mitochondria inducing apoptosis. Thus, we investigated the therapeutic potential of the LIMK1/2 inhibitor CEL_Amide (LIMKi) in FLT3-ITD+ AML. Expression of LIMK1/2 in FLT3-ITD+ cell lines MOLM-13 and MV-4-11 cells could be detected by RT-qPCR and at the protein level. IC50 after LIMKi monotherapy was 440 nM in MOLM-13 cells and 420 nM in MV4-11 cells. Treatment with LIMKi decreased LIMK1 protein levels and repression of inactivating phosphorylation of cofilin in FLT3-ITD+ cells. Combination experiments with LIMKi and FLT3 inhibitors including midostaurin, crenolanib and gilteritinib were synergistic for treatment of MOLM-13 cells while combinations with quizartinib were additive. Combinations of LIMKi and the hypomethylating agent azacitidine or the ROCK inhibitor fasudil were additive. In NOD-SCID mice engrafted with MOLM13-LUC cells, the FLT3 inhibitor midostaurin and LIMKi delayed MOLM13-LUC engraftment as detected by in vivo bioluminescence imaging and the LIMKi and midostaurin combination prolonged significantly survival of leukemic mice. LIMK1/2 inhibition by the small molecule CEL_Amide seems to have promising activity in combination with FLT3 inhibitors in vitro as well as in vivo and may constitute a novel treatment strategy for FLT3-ITD+ AML.
Assuntos
Sinergismo Farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Quinases Lim/antagonistas & inibidores , Mutação , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Amidas/química , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Sequências de Repetição em Tandem , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
LIM kinase 1 (LIMK1) and p21-activated kinase 4 (PAK4) are often over-expressed in breast tumors, which causes aggressive cancer phenotypes and unfavorable clinical outcomes. In addition to the well-defined role in regulating cell division, proliferation and invasion, the two kinases promote activation of the MAPK pathway and cause endocrine resistance through phosphorylating estrogen receptor alpha (ERα). PAK4 specifically phosphorylates LIMK1 and its functional partners, indicating possible value of suppressing both kinases in cancers that over-express PAK4 and/or LIMK1. Here, for the first time, we assessed the impact of combining LIMK1 inhibitor LIMKi 3 and PAK4 inhibitor PF-3758309 in preclinical breast cancer models. LIMK1 and PAK4 pharmacological inhibition synergistically reduced the survival of various cancer cell lines, exhibiting specific efficacy in luminal and HER2-enriched models, and suppressed development and ERα-driven signals in a BT474 xenograft model. In silico analysis demonstrated the cell lines with reliance on LIMK1 were the most prone to be susceptible to PAK4 inhibition. Double LIMK1 and PAK4 targeting therapy can be a successful therapeutic strategy for breast cancer, with a unique efficiency in the subtypes of luminal and HER2-enriched tumors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinases Lim/metabolismo , Pirazóis/administração & dosagem , Pirróis/administração & dosagem , Tiazóis/administração & dosagem , Quinases Ativadas por p21/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinases Lim/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirróis/farmacologia , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/antagonistas & inibidoresRESUMO
Arsenic, an environmental contaminant in drinking water worldwide is well-established to increase cardiovascular diseases (CVDs) in humans. Of these, thrombotic events represent a major adverse effect associated with arsenic exposure, for which an abundance of epidemiological evidence exists. Platelet aggregation constitutes a pivotal step in thrombosis but arsenic alone doesn't induce aggregation and the mechanism underlying arsenic-induced thrombosis still remains unclear. Here we demonstrated that arsenic induces morphological changes of platelets, i.e., contraction and pseudopod projection, the primal events of platelet activation, which can increase platelet reactivity. Arsenite induced prominent platelet shape changes in a dose-dependent manner in freshly isolated human platelets. Of note, arsenite suppressed focal adhesion kinase (FAK) activity, which in turn activated RhoA, leading to altered actin assembly through LIMK activation, and subsequent cofilin inactivation. Arsenic-induced platelet shape change appeared to increase the sensitivity to thrombin and ADP-induced aggregation. Supporting this, latrunculin A, an inhibitor of actin-dynamics abolished it. Taken together, we demonstrated that arsenic induces cytoskeletal changes and shape changes of platelets through FAK-mediated alteration of actin dynamics, which renders platelets reactive to activating stimuli, ultimately contributing to increased thrombosis.
Assuntos
Actinas/metabolismo , Arsenitos/toxicidade , Plaquetas/patologia , Plaquetas/ultraestrutura , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Compostos de Sódio/toxicidade , Difosfato de Adenosina/farmacologia , Adolescente , Adulto , Humanos , Técnicas In Vitro , Quinases Lim/antagonistas & inibidores , Masculino , Selectina-P/biossíntese , Agregação Plaquetária/efeitos dos fármacos , Adulto Jovem , Proteína rhoA de Ligação ao GTPRESUMO
Actin is a highly abundant cytoskeletal protein that is essential for all eukaryotic cells and participates in many structural and functional roles. It has long been noted that estrogen affects cellular morphology. However, recent studies observed that both estrogen and tamoxifen induce a remarkable cytoskeletal remodeling independent of ER. In addition to ER, G protein-coupled estrogen receptor 1 (GPER, also known as GPR30) also binds to estrogen with high affinity and mediates intracellular estrogenic signaling. Here, we show that activation of GPER by its specific agonist G-1 induces re-organization of F-actin cytoskeleton. We further demonstrate that GPER acts through PLCß-PKC and Rho/ROCK-LIMK-Cofilin pathway, which are upstream regulators of F-actin cytoskeleton assembly, thereby enhancing TAZ nuclear localization and activation. Furthermore, we find that LIMK1/2 is critical for GPER activation-induced breast cancer cell migration. Together, our results suggest that GPER mediates G-1-induced cytoskeleton assembly and GPER promotes breast cancer cell migration via PLCß-PKC and Rho/ROCK-LIMK-Cofilin pathway.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Regulação Neoplásica da Expressão Gênica , Quinases Lim/genética , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclopentanos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Quinases Lim/antagonistas & inibidores , Quinases Lim/metabolismo , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
OBJECTIVE: To determine if combined administration of LIMK2 and JNK inhibitors in a rat model of erectile dysfunction induced by cavernosal nerve (CN) injury could restore erectile function by suppressing both cavernosal apoptosis and fibrosis via rectification of molecular pathways related to the structural alterations. METHODS: Sixty 12-week-old male Sprague-Dawley rats were categorized into 4 groups: (1) Sham-surgery (Sham) group, (2) CN-crush-injury (CNCI), (3) CNCI group (CNCI+L+1.0J) treated with a combination of 10.0 mg/kg LIMK2-inhibitors and low-dose (1.0 mg/kg) JNK-inhibitors, and (4) CNCI group (CNCI+L+10.0J) treated with a combination of 10.0 mg/kg LIMK2-inhibitors and a high dose (10.0 mg/kg) of JNK-inhibitors. Ten days after surgery, erectile response, histological-studies, and Western-blot was investigated. RESULTS: The CNCI group showed a reduced maximal ICP/MAP or AUC/MAP, decreased immunohistochemical-staining of α-SMA, decreased SM/collagen ratio, increased phospho-cJun-positive apoptotic cells, increased phospho-LIMK2-positive fibroblasts, increased cJun-phosphorylation, increased LIMK2/Cofilin-phosphorylation, decreased Bcl-2/Bax ratio, and increased protein-expression of fibronectin, compared to the Sham group. Both the CNCI+L+1.0J and CNCI+L+10.0J groups showed improvements in erectile-responses, content of cavernosal α-SMA, number of phospho-cJun-positive apoptotic cells, Bcl-2/Bax ratio and cJun phosphorylation. Their improvements in the CNCI+L+10.0J group showed a tendency to be greater than those in the CNCI+L+1.0J group. Also, in the 2 treatment groups, rectification of SM/collagen ratio, number of phospho-LIMK2-positive fibroblasts, LIMK2/Cofilin-phosphorylation, and protein-expression of fibronectin was observed. CONCLUSION: This study suggests that combined inhibition of JNK and LIMK2 may improve erectile function by suppressing cavernosal apoptosis and fibrosis via restoration of cJun/Bcl-2/Bax and LIMK2/Cofilin pathways at 10 days after CN injury.
Assuntos
Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/etiologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Quinases Lim/antagonistas & inibidores , Pênis/lesões , Pênis/inervação , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Animais , Modelos Animais de Doenças , Combinação de Medicamentos , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Despolimerização de Actina/antagonistas & inibidores , Fatores de Despolimerização de Actina/metabolismo , Animais , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Quinases Lim/antagonistas & inibidores , Quinases Lim/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg-1 LIMK2i and 20.0 mg kg-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.
Assuntos
Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/etiologia , Quinases Lim/antagonistas & inibidores , Traumatismos dos Nervos Periféricos/complicações , Inibidores da Fosfodiesterase 5/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Pressão Arterial , Estimulação Elétrica , Disfunção Erétil/patologia , Masculino , Compressão Nervosa , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/efeitos dos fármacos , Pênis/patologia , Traumatismos dos Nervos Periféricos/patologia , Fosforilação , Pirimidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Sulfonamidas/uso terapêuticoRESUMO
We evaluated whether chronic administration of LIMK2-inhibitors could improve erectile function by alleviating CVOD through suppressing cavernosal fibrosis in a rat model of cavernosal nerve crush-injury (CNCI). Forty-two 12-week-old rats were equally categorized into the three groups: sham-surgery (S), CNCI (I), and CNCI treated with LIMK2-inhibitors (L). The L-group was treated with daily intraperitoneal injection of LIMK2-inhibitors (10.0 mg/kg) for 30-days after surgery. Erectile function was assessed using dynamic-infusion-cavernosometry (DIC). Penile tissue was processed for Masson's-trichrome staining, Western-blotting, and double immunofluorescence. The I-group showed significantly higher maintenance and drop rates as well as lower papaverine response, compared to the S-group. Chronic inhibition of LIMK2 in the L-group significantly improved the DIC parameters compared to those in the I-group, although the parameters were not completely restored to normal control values. Also, the I-group showed a reduced smooth muscle (SM)-to-collagen ratio, decreased immunohistochemical staining for α-SM-actin, increased number of fibroblasts positive for phosphorylated Cofilin, increased LIMK2/Cofilin phosphorylation and increased protein expression of Collagen-1 or Fibronectin, compared to the S-group. The L-group showed significant improvements in SM/collagen ratio and the deposition of Collagen-1 or Fibronectin compared to the I-group, although not completely normalized. According to the densitometry and confocal microscopy results, the L-group showed restoration of LIMK2/Cofilin phosphorylation and amount of fibroblasts positive for phosphorylated Cofilin to the normal control value. In conclusion, chronic inhibition of LIMK2 can improve CVOD and ED by alleviating cavernosal fibrosis via normalizing the LIMK2/Cofilin pathway.
Assuntos
Disfunção Erétil , Quinases Lim , Pênis , Traumatismos dos Nervos Periféricos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/enzimologia , Disfunção Erétil/patologia , Fibrose , Quinases Lim/antagonistas & inibidores , Quinases Lim/metabolismo , Masculino , Pênis/enzimologia , Pênis/patologia , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/enzimologia , Traumatismos dos Nervos Periféricos/patologia , RatosRESUMO
INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) represents an aggressive malignancy often accompanied with a poor prognosis. Owing to the poor mortality and morbidity rates associated with this malignancy, a deeper understanding of the finer molecular changes that occur in ESCC is required in order to identify novel potential targets for early detection and therapy. At present the mechanism by which ESCC functions on a molecular level is not fully understood. Hence, the aim of the present study was to ascertain as to whether microRNA-384 (miR-384) influences the progression of ESCC. MATERIAL AND METHODS: Bioinformatics analysis was initially conducted to identify ESCC-related differentially expressed genes and predict regulatory miRs. After the target relationship between miR-384 and LIMK1 had been verified, the expression of miR-384 and LIMK1 in the EC9706 cell line was altered in an attempt to investigate the regulatory roles of miR-384 in the expression of the LIMK1/cofilin signaling pathway-related genes, cell proliferation, invasion, cell cycle distribution and apoptosis, in addition to lymph node metastasis (LNM) and tumor growth in nude mice. RESULTS: Microarray-based gene expression profiling indicated that miR-384 affected the progression of ESCC through the LIMK1-mediated LIMK1/cofilin signaling pathway. Furthermore, miR-384 and Bax were observed to be poorly expressed, while LIMK1, cofilin and Bcl-2 were highly expressed in ESCC. The obtained evidences indicating that miR-384 targeted and negatively regulated LIMK1. Upregulation of miR-384 or LIMK1 inhibition was determined to block the LIMK1/cofilin signaling pathway, repress cell proliferation, invasion, cell cycle, LNM and tumor growth, while promote cell apoptosis in ESCC. CONCLUSION: Collectively, based on the key findings of the study, miR-384 could sequester LIMK1, which acts to suppress activation of the LIMK1/cofilin signaling pathway, thus ultimately inhibiting the development and progression of ESCC.
Assuntos
Cofilina 1/metabolismo , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Quinases Lim/metabolismo , MicroRNAs/biossíntese , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Cofilina 1/antagonistas & inibidores , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/prevenção & controle , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/prevenção & controle , Feminino , Humanos , Quinases Lim/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ligação Proteica/fisiologia , Ratos , Ratos Nus , Transdução de Sinais/fisiologiaRESUMO
LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM kinases controls cofilin actin-severing activity. Consequently, LIM kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer treatments. LIMK has also been reported to be involved in pathways that are deregulated in hematologic malignancies, with little information regarding cofilin phosphorylation status. We have used proteomic approaches to investigate quantitatively and in detail the phosphorylation status of cofilin in myeloid tumor cell lines of murine and human origin. Our results show that under standard conditions, only a small fraction (10 to 30% depending on the cell line) of cofilin is phosphorylated (including serine 3 phosphorylation). In addition, after a pharmacological inhibition of LIM kinases, a residual cofilin phosphorylation is observed on serine 3. Interestingly, this 2D gel based proteomic study identified new phosphorylation sites on cofilin, such as threonine 63, tyrosine 82 and serine 108.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Carbazóis/farmacologia , Quinases Lim/antagonistas & inibidores , Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Fatores de Despolimerização de Actina/química , Actinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Humanos , Células Mieloides/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
INTRODUCTION: Platelets activation/aggregation with subsequent thrombus formation is the main event in the pathophysiology of acute coronary syndrome. Once activated, platelets show an extensive cytoskeleton rearrangement that leads to recruitment of additional platelets to finally cause haemostatic plug formation. Thus, the cytoskeleton plays a pivotal role in this phenomenon. Colchicine (COLC) is an anti-inflammatory drug proven to reduce major cardiovascular events in patients with coronary artery disease. The molecular mechanisms by which COLC exerts these protective effects remain partially still unknown. Since COLC causes disruption of tubulin, a component of cell cytoskeleton, we investigated whether this drug might interfere with platelet aggregation by acting on cytoskeleton rearrangement. METHODS AND RESULTS: Platelets isolated from healthy volunteers were activated with Adenosine Diphosphate (ADP, 20⯵M) Collagen (COLL, 60⯵g/ml) and Thrombin Activating Receptor Peptide (TRAP 25⯵M) with/without COLC 10⯵M pretreatment. After stimulus, aggregation was measured by light aggregometry overtime. Microtubules structure was assessed by immunohistochemistry and key proteins involved in regulation of actin-filament assembly and contractility such as Myosin Phosphatase Targeting subunit (MYPT), LIM domain kinase 1(LIMK1) and cofilin were evaluated by Western Blot analysis. Colchicine pretreatment significantly blunted ADP/COLL/TRAP-induced platelet aggregation (up to 40%). COLC effects appeared mediated by microtubules depolymerization and cytoskeleton disarrangement associated to inactivation of MYPT and LIMK1 that finally interfered with cofilin activity. CONCLUSIONS: Our data indicate that colchicine exerts anti-platelet effects in vitro via inhibition of key proteins involved in cytoskeleton rearrangement, suggesting that its beneficial cardiovascular properties may be due, at least in part, to an inhibitory effect of platelet activity.
Assuntos
Plaquetas/efeitos dos fármacos , Colchicina/farmacologia , Citoesqueleto/efeitos dos fármacos , Quinases Lim/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Plaquetas/enzimologia , Cofilina 1/metabolismo , Citoesqueleto/enzimologia , Humanos , Quinases Lim/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
LIM kinases are involved in various pathophysiological processes that depend on actin organization. Alteration of microtubule dynamics by LIMK dysregulation is in fact related to tumor progression and metastasis, viral infection, and ocular diseases, such as glaucoma. As a consequence, many efforts have been done in recent years to rationally design small molecules able to inhibit LIMK activity selectively, without affecting other kinases. As a result, compounds optimized in terms of binding affinity and pharmacokinetic parameters have been discovered, that however failed to access clinical trials. In this review, a comprehensive survey of recent LIMK inhibitors is reported, together with SAR considerations and optimization processes.
Assuntos
Desenho de Fármacos , Quinases Lim/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ensaios Clínicos como Assunto , Relação Dose-Resposta a Droga , Humanos , Quinases Lim/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5'-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions. RESULTS: We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy. CONCLUSION: In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Fosfoproteínas Fosfatases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Vitamina B 6/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Meios de Cultura Livres de Soro , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Quinases Lim/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
BACKGROUND AND PURPOSE: In men with benign prostatic hyperplasia, increased smooth muscle tone in the prostate may lead to bladder outlet obstruction and subsequent lower urinary tract symptoms. Consequently, medical treatment aims to inhibit prostate smooth muscle contraction. However, the efficacy of the treatment options available is limited, and improved understanding of mechanisms of prostate smooth muscle contraction and identification of new targets for medical intervention are mandatory. Several studies suggest that LIM kinases (LIMKs) promote smooth muscle contraction; however, this has not yet been examined. Here, we studied effects of the LIMK inhibitors on prostate smooth muscle contraction. EXPERIMENTAL APPROACH: Human prostate tissues were obtained from radical prostatectomy. Phosphorylation of cofilin, a LIMK substrate, was examined using a phospho-specific antibody. Smooth muscle contractions were studied in organ bath experiments. KEY RESULTS: Real-time PCR, Western blot and immunofluorescence suggested LIMKs are expressed in smooth muscle cells of prostate tissues. Two different LIMK inhibitors, SR7826 (1 µM) and LIMKi3 (1 µM), inhibited contractions of prostate strips, which were induced by electrical field stimulation, α1 -adrenoceptor agonists phenylephrine and methoxamine and the TXA2 analogue, U46619. LIMK inhibition in prostate tissues and cultured stromal cells (WPMY-1) was confirmed by cofilin phosphorylation, which was reduced by SR7826 and LIMKi3. In WPMY-1 cells, SR7826 and LIMKi3 caused breakdown of actin filaments and reduced viability. CONCLUSIONS AND IMPLICATIONS: Smooth muscle tone in the hyperplastic human prostate may underlie the effects of LIMKs, which promote contraction. Contraction of prostate strips can be inhibited by small molecule LIMK inhibitors.
Assuntos
Quinases Lim/antagonistas & inibidores , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Tiazóis/farmacologia , Fatores de Despolimerização de Actina/análise , Fatores de Despolimerização de Actina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Estimulação Elétrica , Humanos , Quinases Lim/análise , Quinases Lim/metabolismo , Masculino , Músculo Liso/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/química , Pirazóis/química , Tiazóis/químicaRESUMO
We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg-1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.