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1.
Curr Issues Mol Biol ; 46(10): 11207-11219, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-39451545

RESUMO

Platycladus orientalis is a traditional oriental herbal medicinal plant that is widely used as a component of complex prescriptions for alopecia treatment in Eastern Asia. The effect of PO on hair growth and its underlying mechanism, however, have not been demonstrated or clarified. In this study, we investigated the hair-growth-promoting effect of PO in cultured human dermal papilla cells (hDPCs). Platycladus orientalis leaf extract (POLE) was found to stimulate the proliferation of hDPCs. POLE with higher quercitrin concentration, especially, showed a high level of cellular viability. In the context of cellular senescence, POLE decreased the expression of p16 (CDKN2A) and p21(CDKN1A), which resulted in enhanced proliferation. In addition, growth factor receptors, FGFR1 and VEGFR2/3, and non-receptor tyrosine kinases, ACK1 and HCK, were significantly activated. In addition, LEF1, a transcription factor of Wnt/ß-catenin signaling, was enhanced, but DKK1, an inhibitor of Wnt/ß-catenin signaling, was downregulated by POLE treatment in cultured hDPCs. As a consequence, the expression of growth factors such as bFGF, KGF, and VEGF were also increased by POLE. We further investigated the hair-growth-promoting effect of topically administered POLE over a 12-week period. Our data suggest that POLE could support terminal hair growth by stimulating proliferation of DPCs and that enhanced production of growth factors, especially KGF, occurred as a result of tyrosine kinase ACK1 activation.

2.
Mol Cell Neurosci ; 126: 103865, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37263460

RESUMO

Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease are caused by a progressive and aberrant destruction of neurons in the brain and spinal cord. These disorders lack effective long-term treatments that impact the underlying mechanisms of pathogenesis and as a result, existing options focus primarily on alleviating symptomology. Dysregulated programmed cell death (i.e., apoptosis) is a significant contributor to neurodegeneration, and is controlled by a number of different factors. Rho family GTPases are molecular switches with recognized importance in proper neuronal development and migration that have more recently emerged as central regulators of apoptosis and neuronal survival. Here, we investigated a role for the Rho GTPase family member, Cdc42, and its downstream effectors, in neuronal survival and apoptosis. We initially induced apoptosis in primary cultures of rat cerebellar granule neurons (CGNs) by removing both growth factor-containing serum and depolarizing potassium from the cell medium. We then utilized both chemical inhibitors and adenoviral shRNA targeted to Cdc42 to block the function of Cdc42 or its downstream effectors under either control or apoptotic conditions. Our in vitro studies demonstrate that functional inhibition of Cdc42 or its downstream effector, activated Cdc42-associated tyrosine kinase-1 (ACK-1), had no adverse effects on CGN survival under control conditions, but significantly sensitized neurons to cell death under apoptotic conditions. In conclusion, our results suggest a key pro-survival role for Cdc42/ACK-1 signaling in neurons, particularly in regulating neuronal susceptibility to pro-apoptotic stress such as that observed in neurodegenerative disorders.


Assuntos
Proteínas Tirosina Quinases , Proteínas rho de Ligação ao GTP , Ratos , Animais , Proteínas Tirosina Quinases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/farmacologia , Neurônios/metabolismo , Apoptose/fisiologia
3.
J Biol Chem ; 298(11): 102564, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36206843

RESUMO

The small GTPase CDC42 plays essential roles in neurogenesis and brain development. Previously, we showed that a CDC42 splice variant that has a ubiquitous tissue distribution specifically stimulates the formation of neural progenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neural progenitor cells to neurons. These specific roles of CDC42 and CDC42b in neurogenesis are ascribed to their opposing effects on mTORC1 activity. Specifically, the ubiquitous form of CDC42 stimulates mTORC1 activity and thereby upregulates tissue-specific transcription factors that are essential for neuroprogenitor formation, whereas CDC42b works together with activated CDC42-associated kinase (ACK) to downregulate mTOR expression. Here, we demonstrate that the EGF receptor (EGFR) is an additional and important target of CDC42b and ACK, which is downregulated by their combined actions in promoting neurogenesis. The activation status of the EGFR determines the timing by which neural progenitor cells derived from P19 embryonal carcinoma terminally differentiate into neurons. By promoting EGFR degradation, we found that CDC42b and ACK stimulate autophagy, which protects emerging neurons from apoptosis and helps trigger neural progenitor cells to differentiate into neurons. Moreover, our results reveal that CDC42b is localized in phosphatidylinositol (3,4,5)-triphosphate-enriched microdomains on the plasma membrane, mediated through its polybasic sequence 185KRK187, which is essential for determining its distinct functions. Overall, these findings now highlight a molecular mechanism by which CDC42b and ACK regulate neuronal differentiation and provide new insights into the functional interplay between EGFR degradation and autophagy that occurs during embryonic neurogenesis.


Assuntos
Proteínas Tirosina Quinases , Proteína cdc42 de Ligação ao GTP , Proteínas Tirosina Quinases/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neurogênese , Encéfalo/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
4.
J Biol Chem ; 298(12): 102664, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36334623

RESUMO

Human Tnk1 (thirty-eight negative kinase 1) is a member of the Ack family of nonreceptor tyrosine kinases. Tnk1 contains a sterile alpha motif, a tyrosine kinase catalytic domain, an SH3 (Src homology 3) domain, and a large C-terminal region that contains a ubiquitin association domain. However, specific physiological roles for Tnk1 have not been characterized in depth. Here, we expressed and purified Tnk1 from Sf9 insect cells and established an in vitro assay system using a peptide substrate derived from the Wiskott-Aldrich Syndrome Protein (WASP). By Tnk1 expression in mammalian cells, we found that the N-terminal SAM domain is important for self-association and kinase activity. We also studied a fusion protein, originally discovered in a Hodgkin's Lymphoma cell line, that contains an unrelated sequence from the C17ORF61 gene fused to the C-terminus of Tnk1. Cells expressing the fusion protein showed increased tyrosine phosphorylation of cellular substrates relative to cells expressing WT Tnk1. A truncated Tnk1 construct (residues 1-465) also showed enhanced phosphorylation, indicating that the C17ORF61 sequence was dispensable for the effect. Additionally, in vitro kinase assays with the WASP peptide substrate showed no increase in intrinsic Tnk1 activity in C-terminally truncated constructs, suggesting that the truncations did not simply remove an autoinhibitory element. Fluorescence microscopy experiments demonstrated that the C-terminus of Tnk1 plays an important role in the subcellular localization of the kinase. Taken together, our data suggest that the noncatalytic regions of Tnk1 play important roles in governing activity and substrate phosphorylation.


Assuntos
Proteínas Tirosina Quinases , Domínios de Homologia de src , Humanos , Proteínas Fetais/metabolismo , Mamíferos/metabolismo , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Especificidade por Substrato , Tirosina/metabolismo
5.
Mol Med ; 29(1): 6, 2023 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-36647009

RESUMO

BACKGROUND: Activated Cdc42-associated kinase 1 (ACK1) is a promising druggable target for cancer, but its inhibitors only showed moderate effects in clinical trials. The study aimed to investigate the underlying mechanisms and improve the antitumor efficacy of ACK1 inhibitors. METHODS: RNA-seq was performed to determine the downstream pathways of ACK. Using Lasso Cox regression analysis, we built a risk signature with ACK1-related autophagy genes in the lung adenocarcinoma (LUAD) patients from The Cancer Genome Atlas (TCGA) project. The performance of the signature in predicting the tumor immune environment and response to immunotherapy and chemotherapy were assessed in LUAD. CCK8, mRFP-GFP-LC3 assay, western blot, colony formation, wound healing, and transwell migration assays were conducted to evaluate the effects of the ACK1 inhibitor on lung cancer cells. A subcutaneous NSCLC xenograft model was used for in vivo study. RESULTS: RNA-seq revealed the regulatory role of ACK1 in autophagy. Furthermore, the risk signature separated LUAD patients into low- and high-risk groups with significantly different prognoses. The two groups displayed different tumor immune environments regarding 28 immune cell subsets. The low-risk groups showed high immune scores, high CTLA4 expression levels, high immunophenoscore, and low DNA mismatch repair capacity, suggesting a better response to immunotherapy. This signature also predicted sensitivity to commonly used chemotherapy and targeted drugs. In vitro, the ACK1 inhibitors (AIM-100 and Dasatinib) appeared to trigger adaptive autophagy-like response to protect lung cancer cells from apoptosis and activated the AMPK/mTOR signaling pathway, partially explaining its moderate antitumor efficacy. However, blocking lysosomal degradation with chloroquine/Bafilamycine A1 or inhibiting AMPK signaling with compound C/shPRKAA1 enhanced the ACK1 inhibitor's cytotoxic effects on lung cancer cells. The efficacy of the combined therapy was also verified using a mouse xenograft model. CONCLUSIONS: The resulting signature from ACK1-related autophagy genes robustly predicted survival and drug sensitivity in LUAD. The lysosomal degradation inhibition improved the therapeutic effects of the ACK1 inhibitor, suggesting a potential role for autophagy in therapy evasion.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Proteínas Quinases Ativadas por AMP , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Macrolídeos , Animais , Camundongos
6.
IUBMB Life ; 75(7): 595-608, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36773333

RESUMO

WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is a member of the NEDD4 E3 ubiquitin ligase family. WWP2 ligase activity is regulated by the 2, 3-linker auto-inhibition. Tyrosine phosphorylation of the 2, 3-linker was identified as an activating means for releasing the auto-inhibition of WWP2. However, the tyrosine kinase (TK) for the phosphorylation and activation remains unknown. In this report, we have found that non-receptor TK ACK1 binds to the WW3 domain of WWP2 and phosphorylates WWP2. ACK1 phosphorylates WWP2 at the 2, 3-linker and partially activates the ubiquitination ligase activity. Unexpectedly, tyrosine phosphorylation of the 2, 3-linker seems not a major mode for activation of WWP2, as ACK1 causes much higher activation of the 2, 3-linker tyrosine phosphorylation defective mutants of WWP2 than that of wild-type WWP2. Furthermore, epidermal growth factor (EGF) stimulates tyrosine phosphorylation of WWP2 and this EGF-stimulated phosphorylation of WWP2 is mediated by ACK1. Finally, knockdown of WWP2 by shWWP2 inhibits the EGF-dependent cell proliferation of lung cancer A549 cells, suggesting that WWP2 may function in the EGFR signaling in lung cancer progression. Taken together, our findings have revealed a novel mechanism underlying activation of WWP2.


Assuntos
Neoplasias Pulmonares , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Proteínas Tirosina Quinases/metabolismo , Tirosina/genética
7.
Molecules ; 28(12)2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37375315

RESUMO

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.


Assuntos
Caseína Quinase Idelta , Doenças Neurodegenerativas , Humanos , Regulação para Cima , Doenças Neurodegenerativas/tratamento farmacológico , Receptores Purinérgicos P1/metabolismo , Receptor A2A de Adenosina/metabolismo
8.
Mol Cancer ; 21(1): 138, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35768871

RESUMO

BACKGROUND: Triple-negative breast cancers (TNBCs) are clinically aggressive subtypes of breast cancer. TNBC is difficult to treat with targeted agents due to the lack of commonly targeted therapies within this subtype. Androgen receptor (AR) has been detected in 12-55% of TNBCs. AR stimulates breast tumor growth in the absence of estrogen receptor (ER), and it has become an emerging molecular target in TNBC treatment. METHODS: Ceritinib is a small molecule inhibitor of tyrosine kinase and it is used in the therapy of non-small lung cancer patients. Enzalutamide is a small molecule compound targeting the androgen receptor and it is used to treat prostate cancer. Combination therapy of these drugs were investigated using AR positive breast cancer mouse xenograft models. Also, combination treatment of ceritinib and paclitaxel investigated using AR- and AR low mouse xenograft and patient derived xenograft models. RESULTS: We screened 133 FDA approved drugs that have a therapeutic effect of AR+ TNBC cells. From the screen, we identified two drugs, ceritinib and crizotinib. Since ceritinib has a well- defined role in androgen independent AR signaling pathways, we further investigated the effect of ceritinib. Ceritinib treatment inhibited RTK/ACK/AR pathway and other downstream pathways in AR+ TNBC cells. The combination of ceritinib and enzalutamide showed a robust inhibitory effect on cell growth of AR+ TNBC cells in vitro and in vivo. Interestingly Ceritinib inhibits FAK-YB-1 signaling pathway that leads to paclitaxel resistance in all types of TNBC cells. The combination of paclitaxel and ceritinib showed drastic inhibition of tumor growth compared to a single drug alone. CONCLUSIONS: To improve the response of AR antagonist in AR positive TNBC, we designed a novel combinational strategy comprised of enzalutamide and ceritinib to treat AR+ TNBC tumors through the dual blockade of androgen-dependent and androgen-independent AR signaling pathways. Furthermore, we introduced a novel therapeutic combination of ceritinib and paclitaxel for AR negative or AR-low TNBCs and this combination inhibited tumor growth to a great extent. All agents used in our study are FDA-approved, and thus the proposed combination therapy will likely be useful in the clinic.


Assuntos
Neoplasias de Mama Triplo Negativas , Androgênios/uso terapêutico , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Pirimidinas , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Sulfonas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
9.
BMC Cancer ; 22(1): 84, 2022 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-35057760

RESUMO

Activated Cdc42-associated kinase 1 (ACK1), a kind of tyrosine kinase, is considered to be an oncogene in many cancers, and it is likely to become a potential target for cancer treatment. We found that the expression of the ACK1 gene in colon cancer was higher than that in normal tissues adjacent to cancer, and high expression of the ACK1 gene was associated with poor prognosis of patients. We assessed the prognosis of colon cancer based on ACK1-related genes and constructed a model that can predict the prognosis of colon cancer patients in colon cancer data from The Cancer Genome Atlas (TCGA) database. We then explored the relationship between ACK1 and the immune microenvironment of colon cancer. The overexpression of ACK1 might hinder the function of antigen-presenting cells. The colon cancer prognosis prediction model we constructed has certain significance for clinicians to judge the prognosis of patients with colon cancer. The expression of the ACK1 gene might affect the infiltration level of a variety of immune cells and immunomodulators in the immune microenvironment.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/mortalidade , Proteínas Tirosina Quinases/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Colo/imunologia , Colo/metabolismo , Neoplasias do Colo/imunologia , Bases de Dados Genéticas , Expressão Gênica/genética , Humanos , Inflamação , Valor Preditivo dos Testes , Prognóstico , Transdução de Sinais/genética , Microambiente Tumoral/imunologia
10.
Reprod Domest Anim ; 57(3): 304-313, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34854139

RESUMO

Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.


Assuntos
Doenças dos Bovinos , Infertilidade Masculina , Acetilação , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Metilação de DNA , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/veterinária , Masculino , Espermatogênese/genética , Testículo/metabolismo
11.
Sensors (Basel) ; 22(6)2022 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-35336289

RESUMO

The Internet Engineering Task Force (IETF) has standardized a new framework, called Static Context Header Compression and fragmentation (SCHC), which offers adaptation layer functionality designed to support IPv6 over Low Power Wide Area Networks (LPWANs). The IETF is currently profiling SCHC, and in particular its packet fragmentation and reassembly functionality, for its optimal use over certain LPWAN technologies. Considering the energy constraints of LPWAN devices, it is crucial to determine the energy performance of SCHC packet transfer. In this paper, we present a current and energy consumption model of SCHC packet transfer over Sigfox, a flagship LPWAN technology. The model, which is based on real hardware measurements, allows to determine the impact of several parameters and fragment transmission strategies on the energy performance of SCHC packet transfer over Sigfox. Among other results, we have found that the lifetime of a device powered by a 2000 mAh battery, transmitting packets every 5 days, is 168 days for 2250-byte packets, while it increases to 1464 days for 77-byte packets.

12.
J Biol Chem ; 295(14): 4498-4512, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071086

RESUMO

The small GTPase cell division cycle 42 (CDC42) plays essential roles in neurogenesis and brain development. Previously, using murine embryonic P19 cells as a model system, we showed that CDC42 stimulates mTOR complex 1 (mTORC1) activity and thereby up-regulates transcription factors required for the formation of neural progenitor cells. However, paradoxically, although endogenous CDC42 is required for both the initial transition of undifferentiated P19 cells to neural progenitors and their ultimate terminal differentiation into neurons, ectopic CDC42 overexpression promotes only the first stage of neurogenesis (i.e. the formation of neuroprogenitors) and not the second phase (differentiation into neurons). Here, using both P19 cells and mouse embryonic stem cells, we resolve this paradox, demonstrating that two splice variants of CDC42, differing only in nine amino acid residues in their very C-terminal regions, play distinct roles in neurogenesis. We found that a CDC42 splice variant that has a ubiquitous tissue distribution, termed here as CDC42u, specifically drives the formation of neuroprogenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neuroprogenitor cells to neurons. We further show that the specific roles of CDC42u and CDC42b in neurogenesis are due to their opposing effects on mTORC1 activity. Specifically, CDC42u stimulated mTORC1 activity and thereby induced neuroprogenitor formation, whereas CDC42b worked together with activated CDC42-associated kinase (ACK) in down-regulating mTOR expression and promoting neuronal differentiation. These findings highlight the remarkable functional specificities of two highly similar CDC42 splice variants in regulating distinct stages of neurogenesis.


Assuntos
Neurogênese/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Diferenciação Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética
13.
Mol Cancer ; 19(1): 90, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404161

RESUMO

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond to first-generation EGFR inhibitors; however, the efficacy of these drugs is limited by acquired resistance driven by the EGFR T790M mutation. The discovery of third-generation EGFR inhibitors overcoming EGFR T790M and their new resistance mechanisms have attracted much attention. METHODS: We examined the antitumor activities and potential resistance mechanism of a novel EGFR third-generation inhibitor in vitro and in vivo using ELISA, SRB assay, immunoblotting, flow cytometric analysis, kinase array, qRT-PCR and tumor xenograft models. The clinical effect on a patient was evaluated by computed tomography scan. RESULTS: We identified compound ASK120067 as a novel inhibitor of EGFR T790M, with selectivity over EGFR WT. ASK120067 exhibited potent anti-proliferation activity in tumor cells harboring EGFR T790M (NCI-H1975) and sensitizing mutations (PC-9 and HCC827) while showed moderate or weak inhibition in cells expressing EGFR WT. Oral administration of ASK120067 induced tumor regression in NSCLC xenograft models and in a PDX model harboring EGFR T790M. The treatment of one patient with advanced EGFR T790M-positive NSCLC was described as proof of principle. Moreover, we found that hyperphosphorylation of Ack1 and the subsequent activation of antiapoptotic signaling via the AKT pathway contributed to ASK120067 resistance. Concomitant targeting of EGFR and Ack1 effectively overrode the acquired resistance of ASK120067 both in vitro and in vivo. CONCLUSIONS: Our results idenfity ASK120067 as a promising third-generation EGFR inhibitor and reveal for the first time that Ack1 activation as a novel resistance mechanism to EGFR inhibitors that guide to potential combination strategy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Biochem Soc Trans ; 47(6): 1715-1731, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31845724

RESUMO

Activated Cdc42-associated kinase or ACK, is a non-receptor tyrosine kinase and an effector protein for the small G protein Cdc42. A substantial body of evidence has accumulated in the past few years heavily implicating ACK as a driver of oncogenic processes. Concomitantly, more is also being revealed regarding the signalling pathways involving ACK and molecular details of its modes of action. Some details are also available regarding the regulatory mechanisms of this kinase, including activation and regulation of its catalytic activity, however, a full understanding of these aspects remains elusive. This review considers the current knowledge base concerning ACK and summarizes efforts and future prospects to target ACK therapeutically in cancer.


Assuntos
Neoplasias/terapia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Transporte Biológico , Movimento Celular , Endocitose , Ativação Enzimática , Epigênese Genética , Humanos , Neoplasias/enzimologia , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transativadores/metabolismo
15.
Cell Biol Int ; 42(9): 1097-1105, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28921811

RESUMO

The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células CACO-2 , Proliferação de Células/fisiologia , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/enzimologia , NF-kappa B/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Quinases da Família src/metabolismo
16.
Biochem Biophys Res Commun ; 486(2): 211-217, 2017 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-28189676

RESUMO

The expression levels of the protein tyrosine kinase Ack1 has been reported to be dysregulated in various cancers and involve in oncogenesis and progression. However, the expression and role of Ack1 in osteosarcoma remains unknown. In this study, we found that Ack1 were evidently upregulated in human osteosarcoma tissues and cell lines. In addition, the clinical data showed that high expression level of Ack1 is closely associated with clinical stage and positive distant metastasis, and negatively correlated with overall survival. Then, bioinformatics prediction and luciferase reporter assay indicated Ack1 as a direct target of miR-24, and Ack1 could be downregulated by miR-24 at both the mRNA and protein expression levels. Moreover, Ack1 expression levels were inversely correlated with that of miR-24 in osteosarcoma tissues. Furthermore, functional assay showed that miR-24 significantly suppressed osteosarcoma progression partially mediated by inhibiting Ack1 expression. Finally, western bolt assay revealed that miR-24 regulate AKT/MMPs pathway via Ack1 in osteosarcoma cells. In conclusion, our study demonstrated the suppression of miR-24 on osteosarcoma metastasis by targeting Ack1 via AKT/MMPs pathways, providing a novel strategy for the diagnosis and treatment of osteosarcoma patients.


Assuntos
Neoplasias Ósseas/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , Osteossarcoma/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Mimetismo Molecular , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/mortalidade , Osteossarcoma/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Comput Chem ; 38(15): 1238-1251, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-27782307

RESUMO

Accurate and rapid estimation of relative binding affinities of ligand-protein complexes is a requirement of computational methods for their effective use in rational ligand design. Of the approaches commonly used, free energy perturbation (FEP) methods are considered one of the most accurate, although they require significant computational resources. Accordingly, it is desirable to have alternative methods of similar accuracy but greater computational efficiency to facilitate ligand design. In the present study relative free energies of binding are estimated for one or two non-hydrogen atom changes in compounds targeting the proteins ACK1 and p38 MAP kinase using three methods. The methods include standard FEP, single-step free energy perturbation (SSFEP) and the site-identification by ligand competitive saturation (SILCS) ligand grid free energy (LGFE) approach. Results show the SSFEP and SILCS LGFE methods to be competitive with or better than the FEP results for the studied systems, with SILCS LGFE giving the best agreement with experimental results. This is supported by additional comparisons with published FEP data on p38 MAP kinase inhibitors. While both the SSFEP and SILCS LGFE approaches require a significant upfront computational investment, they offer a 1000-fold computational savings over FEP for calculating the relative affinities of ligand modifications once those pre-computations are complete. An illustrative example of the potential application of these methods in the context of screening large numbers of transformations is presented. Thus, the SSFEP and SILCS LGFE approaches represent viable alternatives for actively driving ligand design during drug discovery and development. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a DNA/química , Desenho de Fármacos , Descoberta de Drogas , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica , Proteínas Quinases p38 Ativadas por Mitógeno/química
18.
Cytotherapy ; 19(2): 311-326, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28088294

RESUMO

BACKGROUND AIMS: Primary hematopoietic stem and progenitor cells (HSPCs) are key components of cell-based therapies for blood disorders and are thus the authentic substrate for related research. We propose that ubiquitous small-volume diagnostic samples represent a readily available and as yet untapped resource of primary patient-derived cells for cell- and gene-therapy studies. METHODS: In the present study we compare isolation and storage methods for HSPCs from normal and thalassemic small-volume blood samples, considering genotype, density-gradient versus lysis-based cell isolation and cryostorage media with different serum contents. Downstream analyses include viability, recovery, differentiation in semi-solid media and performance in liquid cultures and viral transductions. RESULTS: We demonstrate that HSPCs isolated either by ammonium-chloride potassium (ACK)-based lysis or by gradient isolation are suitable for functional analyses in clonogenic assays, high-level HSPC expansion and efficient lentiviral transduction. For cryostorage of cells, gradient isolation is superior to ACK lysis, and cryostorage in freezing media containing 50% fetal bovine serum demonstrated good results across all tested criteria. For assays on freshly isolated cells, ACK lysis performed similar to, and for thalassemic samples better than, gradient isolation, at a fraction of the cost and hands-on time. All isolation and storage methods show considerable variation within sample groups, but this is particularly acute for density gradient isolation of thalassemic samples. DISCUSSION: This study demonstrates the suitability of small-volume blood samples for storage and preclinical studies, opening up the research field of HSPC and gene therapy to any blood diagnostic laboratory with corresponding bioethics approval for experimental use of surplus material.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Separação Celular/métodos , Separação Celular/normas , Terapia Baseada em Transplante de Células e Tecidos/métodos , Leucócitos/patologia , Talassemia/sangue , Preservação de Sangue/métodos , Preservação de Sangue/normas , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Criopreservação , Estudos de Viabilidade , Congelamento , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Contagem de Leucócitos , Leucócitos/fisiologia , Testes Sorológicos , Talassemia/patologia
19.
Sensors (Basel) ; 17(6)2017 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-28604643

RESUMO

Wireless sensor networks (WSNs) are one of the important supporting technologies of edge computing. In WSNs, reliable communications are essential for most applications due to the unreliability of wireless links. In addition, network lifetime is also an important performance metric and needs to be considered in many WSN studies. In the paper, an energy-aware hybrid Automatic Repeat-reQuest protocol (ARQ) scheme is proposed to ensure energy efficiency under the guarantee of network transmission reliability. In the scheme, the source node sends data packets continuously with the correct window size and it does not need to wait for the acknowledgement (ACK) confirmation for each data packet. When the destination receives K data packets, it will return multiple copies of one ACK for confirmation to avoid ACK packet loss. The energy consumption of each node in flat circle network applying the proposed scheme is statistical analyzed and the cases under which it is more energy efficiency than the original scheme is discussed. Moreover, how to select parameters of the scheme is addressed to extend the network lifetime under the constraint of the network reliability. In addition, the energy efficiency of the proposed schemes is evaluated. Simulation results are presented to demonstrate that a node energy consumption reduction could be gained and the network lifetime is prolonged.

20.
J Cell Sci ; 127(Pt 5): 994-1006, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24413169

RESUMO

Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to the development and progression of cancer. Ack1 (activated Cdc42-associated kinase 1, also known as TNK2) is a non-receptor tyrosine kinase that has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet its precise functions remain elusive. In this report, we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon stimulation with EGF. These structures are proposed to be the isolation membranes that arise during formation of autophagosomes. In addition, we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, through a ubiquitin-associated domain, and this interaction decreases upon treatment with EGF, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, knockdown of Ack1 results in accelerated localisation of EGFR to lysosomes upon treatment with EGF. Structure-function analyses of a panel of Ack1 deletion mutants revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin-binding domain both contribute to colocalisation with EGFR, whereas the UBA domain is essential for colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores ErbB/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas/metabolismo , Autofagia , Fator de Crescimento Epidérmico/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fagossomos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteína Sequestossoma-1 , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/metabolismo
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