RESUMO
Breast cancer is the most common malignancy accounting for 12.5% of all newly diagnosed cancer cases across the globe. Breast cancer cells are known to metastasize to distant organs (i.e., brain), wherein they can exhibit a dormant phenotype for extended time periods. These dormant cancer cells exhibit reduced proliferation and therapeutic resistance. However, the mechanisms by which dormant cancer cells exhibit resistance to therapy, in the context of brain metastatic breast cancer (BMBC), is not well understood. Herein, we utilized hyaluronic acid (HA) hydrogels with varying stiffnesses to study drug responsiveness in dormant vs. proliferative BMBC cells. It was found that cells cultured on soft HA hydrogels (â¼0.4 kPa) that showed a non-proliferative (dormant) phenotype exhibited resistance to Paclitaxel or Lapatinib. In contrast, cells cultured on stiff HA hydrogels (â¼4.5 kPa) that showed a proliferative phenotype exhibited responsiveness to Paclitaxel or Lapatinib. Moreover, dormancy-associated resistance was found to be due to upregulation of the serum/glucocorticoid regulated kinase 1 (SGK1) gene which was mediated, in part, by the p38 signaling pathway. Accordingly, SGK1 inhibition resulted in a dormant-to-proliferative switch and response to therapy. Overall, our study demonstrates that matrix stiffness influences dormancy-associated therapy response mediated, in part, via the p38/SGK1 axis.
Assuntos
Neoplasias Encefálicas , Neoplasias da Mama , Proliferação de Células , Ácido Hialurônico , Hidrogéis , Lapatinib , Paclitaxel , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Hidrogéis/química , Hidrogéis/farmacologia , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/patologia , Lapatinib/farmacologia , Lapatinib/administração & dosagem , Paclitaxel/farmacologia , Paclitaxel/química , Paclitaxel/administração & dosagem , Feminino , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidoresRESUMO
The serine/threonine kinase SGK1 is an activator of the ß-catenin pathway and a powerful stimulator of cartilage degradation that is found to be upregulated under genomic control in diseased osteoarthritic cartilage. Today, no oral disease-modifying treatments are available and chronic treatment in this indication sets high requirements for the drug selectivity, pharmacokinetic, and safety profile. We describe the identification of a highly selective druglike 1H-pyrazolo[3,4-d]pyrimidine SGK1 inhibitor 17a that matches both safety and pharmacokinetic requirements for oral dosing. Rational compound design was facilitated by a novel hSGK1 co-crystal structure, and multiple ligand-based computer models were applied to guide the chemical optimization of the compound ADMET and selectivity profiles. Compounds were selected for subchronic proof of mechanism studies in the mouse femoral head cartilage explant model, and compound 17a emerged as a druglike SGK1 inhibitor, with a highly optimized profile suitable for oral dosing as a novel, potentially disease-modifying agent for osteoarthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Modelos Animais de Doenças , Proteínas Imediatamente Precoces/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Animais , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/enzimologia , Osteoartrite/patologia , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-DawleyRESUMO
Cardiac hypertrophy is a pivotal pathophysiological step of various cardiovascular diseases, which eventually leads to heart failure and death. Extracts of Rhodiola species (Ext.R), a class of commonly used medicinal herbs in Europe and East Asia, can attenuate cardiac hypertrophy both in vitro and in vivo. Serum/glucocorticoid regulated kinase 1 (SGK1) is identified as a potential target of Ext. R. By mass spectrometry-based kinase inhibitory assay, herbacetin (HBT) from Ext.R is identified as a novel SGK1 inhibitor with IC50 of 752 nmol. Thermal shift assay, KINOMEscan in vitro assay combined with molecular docking proves a direct binding between HBT and SGK1. Site-specific mutation of Asp177 in SGK1 completely ablates the inhibitory activity of HBT. The presence of OH groups at the C-3, C-8, C-4' positions of flavonoids is suggested to be favorable for the inhibition of SGK1 activity. Finally, HBT significantly suppresses cardiomyocyte hypertrophy in vitro and in vivo, reduces reactive oxygen species (ROS) synthesis and calcium accumulation. HBT decreases phosphorylation of SGK1 and regulates its downstream forkhead box protein O1 (FoxO1) signaling pathway. Taken together, the findings suggest that a panel of flavonoids structurally related to HBT may be novel leads for developing new therapeutics against cardiac hypertrophy.
Assuntos
Cardiomegalia/tratamento farmacológico , Flavonoides/farmacologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Cardiomegalia/genética , Células Cultivadas , Modelos Animais de Doenças , Proteínas Imediatamente Precoces/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
BACKGROUND: The serum and glucocorticoid-induced kinase-1 (SGK1) belonging to the AGC protein kinase family phosphorylates serine and threonine residues of target proteins. It regulates numerous ion channels and transporters and promotes survival under cellular stress. Unique to SGK1 is a tight control at transcriptional and post-transcriptional levels. SGK1 regulates multiple signal transduction pathways related to tumor development. Several studies have reported that SGK1 is upregulated in different types of human malignancies and induces resistance against inhibitors, drugs, and targeted therapies. RESULTS AND CONCLUSION: This review highlights the cellular functions of SGK1, its crucial role in cancer development, and clinical insights for SGK1 targeted therapies. Furthermore, the role of SGK1-mediated autophagy as a potential therapeutic target for cancer has been discussed.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Carcinogênese/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Neoplasias/diagnóstico , Neoplasias/genética , Oncogenes , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ativação Transcricional , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to its ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6ß (wt) and dlx3.1-6ß (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter:lacz expression cassette. A ≥10-fold difference in ß-galactosidase activity was observed in cells infected with KOS6ß compared to dlx3.1-6ß, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z'-factor score of ≥0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.
Assuntos
Herpesvirus Humano 1/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Ativação Transcricional/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Coleta de Dados , Expressão Gênica , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Transcricional/genéticaRESUMO
Liver cancer most commonly develops in patients with chronic liver disease, the etiology of which includes viral hepatitis (B and C), alcohol, obesity, dietary carcinogens, and so forth. The current treatment modalities, including surgical resection and liver transplantation, have been found far from effective. Hence, there is an obvious critical need to develop alternative strategies for the treatment of it. In this review, we discuss the formation process and therapeutic targets of liver cancer. Currently, targeted therapy is limited to sorafenib, lenvatinib, regorafenib, ramucirumab and cabozantinib which leads to a survival benefit in patients, but on the other hand is hampered by the occurrence of drug resistance. Pleasingly and importantly, there are multiple natural products undergoing clinical evaluation in liver cancer, such as polyphenols like icaritin, resveratrol, and silybin, saponins including ginsenoside Rg3 and glycyrrhizinate, alkaloid containing irinotecan and berberine and inorganic compound arsenic trioxide at present. Preclinical and clinical studies have shown that these compounds inhibit liver cancer formation owing to the influence on the anti-viral, anti-inflammation, anti-oxidant, anti-angiogenesis and anti-metastasis activity. Furthermore, a series of small molecule derivatives inspired by the aforementioned compounds are designed and synthesized according to structure-activity relationship studies. Drug combination and novel type of drug-targeted delivery system thereof have been well developed. This article is ended by a perspective remark of futuristic development of natural product-based therapeutic regimen for liver cancer treatment. We expect that this review is an account for current status of natural products as promising anti-liver cancer treatments and should contribute to its understanding.
Assuntos
Antineoplásicos/uso terapêutico , Produtos Biológicos/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Sorafenibe/uso terapêutico , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Ginsenosídeos/uso terapêutico , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Resveratrol/uso terapêuticoRESUMO
Blood-brain barrier (BBB) disruption is one of the most important pathological changes following cerebral ischemia-reperfusion. We tested whether inhibition of the serum and glucocorticoid regulated kinase 1 (SGK1) would decrease BBB disruption and contribute to decreasing infarct size in the first few hours of cerebral ischemia-reperfusion within the thrombolysis therapy time window. After transient middle cerebral artery occlusion (MCAO), an SGK1 inhibitor GSK650394, or vehicle was administered into the lateral ventricle of rats. After one hour of MCAO and two hours of reperfusion, we determined BBB disruption using the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid, and also determined infarct size, phosphorylation of NDRG1, and MMP2 protein level. Ischemia-reperfusion increased (+34%, p < 0.05) and GSK650394 decreased (-25%, p < 0.05) the Ki in the ischemic-reperfused cortex. GSK650394 decreased the percentage of cortical infarct (-31%, p < 0.001). At the same time GSK650394 reduced NDRG1 phosphorylation and MMP2 protein level in the ischemic-reperfused cortex suggesting that SGK1 was inhibited by GSK650394 and that lower MMP2 could be one of the mechanisms of decreased BBB disruption. Collectively our data suggest that GSK650394 could be neuroprotective and one of the mechanisms of the neuroprotection could be decreased BBB disruption. SGK1 inhibition within the thrombolysis therapy time window might reduce cerebral ischemia-reperfusion injury.
Assuntos
Benzoatos/farmacologia , Barreira Hematoencefálica/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Infarto Cerebral/patologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Traumatismo por Reperfusão/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. But the drawback is the low water solubility which prevents its further development. In this context, we successfully overcame this limitation by synthesizing two novel prodrugs introducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue distribution confirmed a better profile highlighting the importance of the prodrug approach. Finally, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Fibrinolisina/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Pró-Fármacos/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estabilidade de Medicamentos , Feminino , Meia-Vida , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Transplante HeterólogoRESUMO
It has been reported that the gut microbiome modulates postoperative cognitive dysfunction (POCD), and that administration of probiotics (VSL#3) may effectively relieve POCD. In this study, we aimed to identify the underlying mechanism of VSL#3 in POCD. A mouse model of POCD was constructed in adult male C57BL/6 mice, which were then treated with VSL#3. VSL#3 exerted a protective role against POCD and resultant neuronal apoptosis. The expression of miR-146a was found to be downregulated in hippocampal tissues of POCD mice, while VSL#3 could restore its expression. Loss- and gain-function approaches were conducted to determine the roles of microRNA (miR)-146a, B-cell translocation gene 2 (BTG2), and Bcl-2-associated X protein (Bax) in post-operative effects on cognitive function and neuronal apoptosis. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured to determine oxidative stress in brain tissue. The dual-luciferase reporter gene assay identified that miR-146a could target BTG2 and negatively regulate its expression. BTG2 knockdown suppressed neuronal apoptosis and contributed to shortened time of latency, prolonged time of mice spent in the target quadrant, and reduced oxidative stress through downregulating Bax expression. Finally, VSL#3 treatment upregulated the expression of miR-146a to block BTG2/Bax axis and consequently inhibited neuronal apoptosis and reduced oxidative stress in POCD mice. Taken together, the study suggested that miR-146a-mediated suppression of BTG2/Bax contributed to the protective role of probiotics treatment against POCD.
Assuntos
Proteínas Imediatamente Precoces/biossíntese , MicroRNAs/biossíntese , Complicações Cognitivas Pós-Operatórias/dietoterapia , Complicações Cognitivas Pós-Operatórias/metabolismo , Probióticos/administração & dosagem , Proteínas Supressoras de Tumor/biossíntese , Proteína X Associada a bcl-2/biossíntese , Animais , Linhagem Celular , Expressão Gênica , Proteínas Imediatamente Precoces/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complicações Cognitivas Pós-Operatórias/prevenção & controle , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteína X Associada a bcl-2/antagonistas & inibidoresRESUMO
The serum- and glucocorticoid-regulated kinase (SGK) family consists of three isoforms (SGK1, SGK2, and SGK3) that have been implicated in the regulation of tumor growth, metastasis, autophagy, and epithelial ion transport. SGK1 and SGK3 play essential roles in protein kinase B (AKT or PKB)-independent phosphoinositide 3-kinases (PI3K)-mediated tumorigenesis, as evidenced by the significantly elevated expression levels of SGK1 and SGK3 in many cancers, including prostate cancer, colorectal carcinoma, estrogen-dependent breast cancer, and glioblastoma. Therefore, SGK is a potential target for anticancer therapy. A small kinase-focused library comprising 160 compounds was screened against SGK1 using a fluorescence polarization-based kinase assay that yielded a Z'-factor of 0.82. Among the 39 compounds obtained as initial hits in a primary screen, 12 compounds contained the thiazolidine-2,4-dione scaffold. The inhibitory mechanisms of the most potent hit, KMU010402, were further investigated using kinetic analyses, followed by determination of the inhibition constants for SGK1, SGK2, and SGK3. Molecular modeling was used to propose a potential binding mode of KMU010402 to SGK1.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Polarização de Fluorescência/métodos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Descoberta de Drogas/métodos , Humanos , Cinética , Modelos Moleculares , Isoformas de Proteínas , Relação Estrutura-AtividadeRESUMO
Vascular calcification may result from stimulation of osteogenic signalling with upregulation of the transcription factors CBFA1, MSX2 and SOX9, as well as alkaline phosphatase (ALPL), which degrades and thus inactivates the calcification inhibitor pyrophosphate. Osteogenic signalling further involves upregulation of the Ca2+-channel ORAI1. The channel is activated by STIM1 and then accomplishes store-operated Ca2+ entry. ORAI1 and STIM1 are upregulated by the serum & glucocorticoid inducible kinase 1 (SGK1) which is critically important for osteogenic signalling. Stimulators of vascular calcification include vasopressin. The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to vasopressin upregulates ORAI1 and/or STIM1 expression, store-operated Ca2+ entry and osteogenic signalling. To this end, HAoSMCs were exposed to vasopressin (100 nM, 24 h) without or with additional exposure to ORAI1 blocker MRS1845 (10 µM) or SGK1 inhibitor GSK-650394 (1 µM). Transcript levels were measured using q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and store-operated Ca2+ entry from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, vasopressin enhanced the transcript levels of ORAI1 and STIM1, store-operated Ca2+ entry, as well as the transcript levels of CBFA1, MSX2, SOX9 and ALPL. The effect of vasopressin on store-operated Ca2+ entry as well as on transcript levels of CBFA1, MSX2, SOX9 and ALPL was virtually abrogated by MRS1845 and GSK-650394. In conclusion, vasopressin stimulates expression of ORAI1/STIM1, thus augmenting store-operated Ca2+ entry and osteogenic signalling. In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394. KEY MESSAGES: ⢠In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. ⢠VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). ⢠VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. ⢠VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Proteína ORAI1/biossíntese , Calcificação Vascular/metabolismo , Vasopressinas/farmacologia , Aorta/citologia , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Nitrendipino/análogos & derivados , Nitrendipino/farmacologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Osteogênese/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Molécula 1 de Interação Estromal/biossíntese , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/fisiologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Calcificação Vascular/prevenção & controleRESUMO
Diurnal variations in pain hypersensitivity are common in chronic pain disorders. Temporal exacerbation of neuropathic pain hypersensitivity is dependent on diurnal variations in glucocorticoid secretion from the adrenal glands. We previously demonstrated that spinal expression of serum- and glucocorticoid-inducible kinase-1 (SGK-1) is associated with glucocorticoid- induced exacerbation of pain hypersensitivity, but there are no available strategies to inhibit SGK-1 in the spinal cord. By screening a clinically approved drug library (more than 1,200 drugs), we found that sulfasalazine (SSZ) has inhibitory effects on SGK-1. SSZ is a prodrug composed of 5-aminosalicylic acid and sulfapyridine linked by NN bond, which is therapeutically effective for inflammatory bowel diseases. However, the NN bond in SSZ was necessary for its inhibitory action against SGK-1. Although intrathecal injection of SSZ to nerve-injured mice significantly alleviated mechanical pain hypersensitivity, no significant anti- neuropathic pain effects of SSZ were detected after oral administration due to its low bioavailability and limited spinal distribution, which were associated with efflux by the xenobiotic transporter breast cancer resistance protein (BCRP). Concomitant oral administration of SSZ with febuxostat (FBX), which is an approved drug to inhibit BCRP, improved the distribution of SSZ to the spinal cord. The concomitant oral administration with FBX also increased the anti-neuropathic pain effects of SSZ. Our study revealed a previously unrecognized pharmacological effect of SSZ to alleviate SGK-1-induced painful peripheral neuropathy, and concomitant oral administration of SSZ with FBX may also be a preventative option for diurnal exacerbation of neuropathic pain hypersensitivity.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Proteínas Imediatamente Precoces/antagonistas & inibidores , Neuralgia/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Sulfassalazina/administração & dosagem , Animais , Benzoatos/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Humanos , Proteínas Imediatamente Precoces/metabolismo , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Neuralgia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Medula Espinal/metabolismoRESUMO
At present, chronic post-surgical pain (CPSP) is difficult to prevent and cure clinically because of our lack of understanding of its mechanisms. Surgical injury induces the upregulation of voltage-gated sodium channel Nav1.7 in dorsal root ganglion (DRG) neurons, suggesting that Nav1.7 is involved in the development of CPSP. However, the mechanism leading to persistent dysregulation of Nav1.7 is largely unknown. Given that nerve growth factor (NGF) induces a long-term increase in the neuronal hyperexcitability after injury, we hypothesized that NGF might cause the long-term dysregulation of Nav1.7. In this study, we aimed to investigate whether Nav1.7 regulation by NGF is involved in CPSP and thus contributes to the specific mechanisms involved in the development of CPSP. Using conditional nociceptor-specific Nav1.7 knockout mice, we confirmed the involvement of Nav1.7 in NGF-induced pain and identified its role in the maintenance of pain behavior during long-term observations (up to 14 days). Using western blot analyses and immunostaining, we showed that NGF could trigger the upregulation of Nav1.7 expression and thus support the development of CPSP in rats. Using pharmacological approaches, we showed that the increase of Nav1.7 might be partly regulated by an NGF/TrkA-SGK1-Nedd4-2-mediated pathway. Furthermore, reversing the upregulation of Nav1.7 in DRG could alleviate spinal sensitization. Our results suggest that the maintained upregulation of Nav1.7 triggered by NGF contributes to the development of CPSP. Attenuating the dysregulation of Nav1.7 in peripheral nociceptors may be a strategy to prevent the transition from acute post-surgical pain to CPSP.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fator de Crescimento Neural/farmacologia , Dor Pós-Operatória/genética , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima , Analgésicos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Benzamidas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hidrazinas/farmacologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Indóis/farmacologia , Masculino , Camundongos Knockout , Modelos Biológicos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor Pós-Operatória/patologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos Sprague-Dawley , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Medula Espinal/patologia , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
In diabetes mellitus, hyperglycemia promotes the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) to enhance medial vascular calcification, a common complication strongly associated with cardiovascular disease and mortality. The mechanisms involved are, however, still poorly understood. Therefore, the present study explored the potential role of serum- and glucocorticoid-inducible kinase 1 (SGK1) during vascular calcification promoted by hyperglycemic conditions. Exposure to high-glucose conditions up-regulated the SGK1 expression in primary human aortic VSMCs. High glucose increased osteogenic marker expression and activity and, thus, promoted the osteogenic transdifferentiation of VSMCs, effects significantly suppressed by additional treatment with the SGK1 inhibitor EMD638683. Moreover, high glucose augmented the mineralization of VSMCs in the presence of calcification medium, effects again significantly reduced by SGK1 inhibition. Similarly, SGK1 knockdown blunted the high glucose-induced osteogenic transdifferentiation of VSMCs. The osteoinductive signaling promoted by high glucose required SGK1-dependent NF-kB activation. In addition, advanced glycation end products (AGEs) increased the SGK1 expression in VSMCs, and SGK1 inhibition was able to interfere with AGEs-induced osteogenic signaling. In conclusion, SGK1 is up-regulated and mediates, at least partly, the osteogenic transdifferentiation and calcification of VSMCs during hyperglycemic conditions. Thus, SGK1 inhibition may reduce the development of vascular calcification promoted by hyperglycemia in diabetes.
Assuntos
Calcinose/genética , Diabetes Mellitus/genética , Hiperglicemia/genética , Proteínas Imediatamente Precoces/genética , Proteínas Serina-Treonina Quinases/genética , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Aorta/patologia , Benzamidas/farmacologia , Calcinose/metabolismo , Calcinose/patologia , Transdiferenciação Celular/genética , Diabetes Mellitus/patologia , Glucose/efeitos adversos , Produtos Finais de Glicação Avançada/genética , Humanos , Hidrazinas/farmacologia , Hiperglicemia/patologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Osteogênese/genética , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/genéticaRESUMO
Cardiopulmonary arrest (CA) is the leading cause of death and disability in the United States. CA-induced brain injury is influenced by multifactorial processes, including reduced cerebral blood flow (hypoperfusion) and neuroinflammation, which can lead to neuronal cell death and functional deficits. We have identified serum and glucocorticoid-regulated kinase-1 (SGK1) as a new target in brain ischemia previously described in the heart, liver, and kidneys (i.e., diabetes and hypertension). Our data suggest brain SGK1 mRNA and protein expression (i.e., hippocampus), presented with hypoperfusion (low cerebral blood flow) and neuroinflammation, leading to further studies of the potential role of SGK1 in CA-induced brain injury. We used a 6-min asphyxia cardiac arrest (ACA) rat model to induce global cerebral ischemia. Modulation of SGK1 was implemented via GSK650394, a SGK1-specific inhibitor (1.2 µg/kg icv). Accordingly, treatment with GSK650394 attenuated cortical hypoperfusion and neuroinflammation (via Iba1 expression) after ACA, whereas neuronal survival was enhanced in the CA1 region of the hippocampus. Learning/memory deficits were observed 3 days after ACA but ameliorated with GSK650394. In conclusion, SGK1 is a major contributor to ACA-induced brain injury and neurological deficits, while inhibition of SGK1 with GSK650394 provided neuroprotection against CA-induced hypoperfusion, neuroinflammation, neuronal cell death, and learning/memory deficits. Our studies could lead to a novel, therapeutic target for alleviating brain injury following cerebral ischemia.NEW & NOTEWORTHY Upregulation of SGK1 exacerbates brain injury during cerebral ischemia. Inhibition of SGK1 affords neuroprotection against cardiac arrest-induced hypoperfusion, neuroinflammation, neuronal cell death, and neurological deficits.
Assuntos
Lesões Encefálicas/metabolismo , Parada Cardíaca/complicações , Proteínas Imediatamente Precoces/genética , Memória , Proteínas Serina-Treonina Quinases/genética , Animais , Benzoatos/farmacologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/etiologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Circulação Cerebrovascular , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
For many tumor types chemotherapy still represents the therapy of choice and many standard treatments are based on the use of platinum (PT) drugs. However, de novo or acquired resistance to platinum is frequent and leads to disease progression. In Epithelial Ovarian Cancer (EOC) patients, PT-resistant recurrences are very common and improving the response to treatment still represents an unmet clinical need. To identify new modulators of PT-sensitivity, we performed a loss-of-function screening targeting 680 genes potentially involved in the response of EOC cells to platinum. We found that SGK2 (Serum-and Glucocorticoid-inducible kinase 2) plays a key role in PT-response. We show here that EOC cells relay on the induction of autophagy to escape PT-induced death and that SGK2 inhibition increases PT sensitivity inducing a block in the autophagy cascade due to the impairment of lysosomal acidification. Mechanistically we demonstrate that SGK2 controls autophagy in a kinase-dependent manner by binding and inhibiting the V-ATPase proton pump. Accordingly, SGK2 phosphorylates the subunit V1H (ATP6V1H) of V-ATPase and silencing or chemical inhibition of SGK2, affects the normal autophagic flux and sensitizes EOC cells to platinum. Hence, we identified a new pathway that links autophagy to the survival of cancer cells under platinum treatment in which the druggable kinase SGK2 plays a central role. Our data suggest that blocking autophagy via SGK2 inhibition could represent a novel therapeutic strategy to improve patients' response to platinum.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Epitelial do Ovário/tratamento farmacológico , Proteínas Imediatamente Precoces/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Alzheimer's disease (AD), which is characterized by impairment of cognitive functions, is a chronic neurodegenerative disease that mainly affects the elderly. Currently available anti-AD drugs can only offer limited symptom-relieving effects. "One-compound-Multitargeted Strategy" have been recognized as the promising way to win the war against AD. Herein we report a potential anti-AD agent PT109 with multi-functions. First, an 81-kinase screening was carried out and results showed that PT109 potently inhibited c-Jun N-terminal kinases and Serum and glucocorticoid-inducible kinase 1, which are the important signaling molecules involved in neurogenesis, neuroprotection and neuroinflammation and mildly inhibit glycogen synthase kinase-3ß as well as protein kinase C gamma, both are involved in AD pathological processes. In addition, invitro studies of immunofluorescent staining and Western blot showed that PT109 might promote the neurogenesis of C17.2 cells and induce synaptogenesis in primary cultured rat hippocampal neurons. We detected and confirmed the neuroprotective effect of PT109 in cultured HT22 cells by MTT assay, dehydrogenase assay, glutathione assay and reactive oxygen species assay. Furthermore, the results of Western blot, ELISA assay and immunofluorescent staining indicated that PT109 attenuated lipopolysaccharide-induced inflammation in BV2 cells and primary astrocytes. The results of Morris water maze and Step-through test indicated that PT109 improved the spatial learning ability in APP/PS1 mice. More importantly, the invivo pharmacokinetic parameters indicated that PT109 had better medicinal properties. Taken together, our findings suggest that PT109 may be a promising candidate for treating AD through multiple targets although further studies are ought to be conducted.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Descoberta de Drogas , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Terapia de Alvo Molecular , Teste do Labirinto Aquático de Morris/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacocinética , Presenilina-1/genética , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
A lower mortality rate is observed in obese patients with acute lung injury (ALI), which is referred to as the obesity paradox, in several studies and recent meta-analyses. Hyperinsulinemia is characterized as the primary effect of obesity, and exogenous insulin attenuates LPS-induced pulmonary edema. The detailed mechanism responsible for the effect of hyperinsulinemia on pulmonary edema and alveolar filling needs to be elucidated. SD rats were fed with a high-fat diet (HFD) for a total of 14 weeks. SD rats were anesthetized and intraperitoneally injected with 10 mg/kg lipopolysaccharide (LPS), while control rats received only saline vehicle. Insulin receptor antagonist S961 (20 nmol/kg) was given by the tail vein and serum, and glucocorticoid-induced protein kinase-1 (SGK-1) inhibitor EMD638683 (20 mg/kg) was administrated intragastrically prior to LPS exposure. The lungs were isolated for the measurement of alveolar fluid clearance. The protein expression of epithelial sodium channel (ENaC) was detected by Western blot. Insulin level in serum was significantly higher in HFD rats compared with normal diet rats in the presence or absence of LPS pretreatment. Hyperinsulinemia induced by high fat feeding increased alveolar fluid clearance and the abundance of α-ENaC, ß-ENaC, and γ-ENaC in both normal rats and ALI rats. Moreover, these effects were reversed in response to S961. EMD638683 prevented the simulation of alveolar fluid clearance and protein expression of ENaC in HFD rats with ALI. These findings suggest that hyperinsulinemia induced by obesity results in the stimulation of alveolar fluid clearance via the upregulation of the abundance of ENaC in clinical acute lung injury, whereas theses effects are prevented by an SGK-1 inhibitor.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Canais Epiteliais de Sódio/metabolismo , Hiperinsulinismo/metabolismo , Obesidade/metabolismo , Edema Pulmonar/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Benzamidas/farmacologia , Dieta Hiperlipídica , Canais Epiteliais de Sódio/efeitos dos fármacos , Hidrazinas/farmacologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RatosRESUMO
Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na+/K+-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na+/K+ pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na+/K+ pump capacity was estimated from K+-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na+/K+ pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (Vm) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on Vm was virtually abrogated by ouabain. Na+/K+ α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na+/K+ pump capacity.
Assuntos
Membrana Celular/patologia , Neuroacantocitose/metabolismo , Neuroacantocitose/patologia , Neurônios/patologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Estudos de Casos e Controles , Diferenciação Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/citologia , Lítio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND: Radiosensitivity is limited in cervical cancer (CC) patients due to acquired radiation resistance. In our previous studies, we found that immediate-early response 5 (IER5) is upregulated in CC cells upon radiation exposure and decreases cell survival by promoting apoptosis. The details on the transcriptional regulation of radiation-induced IER5 expression are unknown. Studies in recent years have suggested that Pol II-associated factor 1 (PAF1) is a pivotal transcription factor for certain genes "induced" during tumor progression. In this study, we investigated the role of PAF1 in regulating IER5 expression during CC radiotherapy. METHODS: PAF1 expression in CC cells was measured by western blotting, immunohistochemistry, and qRT-PCR, and the localization of PAF1 and IER5 was determined by immunofluorescence. The effect of PAF1 and IER5 knockdown by siRNA in Siha and Hela cells was studied by western blotting, qRT-PCR, CCK-8 assay, and flow cytometry. The physical interaction of PAF1 with the IER5 promoter and enhancers was confirmed using chromatin immunoprecipitation and qPCR with or without enhancers knockout by CRISPR/Cas9. RESULTS: We confirmed that PAF1 was highly expressed in CC cells and that relatively low expression of IER5 was observed in cells with highly expressed PAF1 in the nucleus. PAF1 knockdown in Siha and Hela cells was associated with increased expression of IER5, reduced cell viability and higher apoptosis rate in response to radiation exposure, while simultaneous PAF1 and IER5 knockdown had little effect on the proportion of apoptotic cells. We also found that PAF1 hindered the transcription of IER5 by promoting Pol II pausing at the promoter-proximal region, which was primarily due to the binding of PAF1 at the enhancers. CONCLUSIONS: PAF1 reduces CC radiosensitivity by inhibiting IER5 transcription, at least in part by regulating its enhancers. PAF1 might be a potential therapeutic target for overcoming radiation resistance in CC patients.