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1.
Exp Neurobiol ; 30(5): 319-328, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34737237

RESUMO

The TMEM43 has been studied in human diseases such as arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5) and auditory neuropathy spectrum disorder (ANSD). In the heart, the p.(Ser358Leu) mutation has been shown to alter intercalated disc protein function and disturb beating rhythms. In the cochlea, the p.(Arg372Ter) mutation has been shown to disrupt connexin-linked function in glia-like supporting cells (GLSs), which maintain inner ear homeostasis for hearing. The TMEM43-p.(Arg372Ter) mutant knock-in mice displayed a significantly reduced passive conductance current in the cochlear GLSs, raising a possibility that TMEM43 is essential for mediating the passive conductance current in GLSs. In the brain, the two-pore-domain potassium (K2P) channels are generally known as the "leak channels" to mediate background conductance current, raising another possibility that K2P channels might contribute to the passive conductance current in GLSs. However, the possible association between TMEM43 and K2P channels has not been investigated yet. In this study, we examined whether TMEM43 physically interacts with one of the K2P channels in the cochlea, KCNK3 (TASK-1). Utilizing co-immunoprecipitation (IP) assay and Duolink proximity ligation assay (PLA), we revealed that TMEM43 and TASK-1 proteins could directly interact. Genetic modifications further delineated that the intracellular loop domain of TMEM43 is responsible for TASK-1 binding. In the end, gene-silencing of Task-1 resulted in significantly reduced passive conductance current in GLSs. Together, our findings demonstrate that TMEM43 and TASK-1 form a protein-protein interaction in the cochlea and provide the possibility that TASK-1 is a potential contributor to the passive conductance current in GLSs.

2.
J Enzyme Inhib Med Chem ; 36(1): 2016-2024, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34514924

RESUMO

Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.


Assuntos
4-Aminobutirato Transaminase/biossíntese , 4-Aminobutirato Transaminase/isolamento & purificação , 4-Aminobutirato Transaminase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
3.
Mol Oncol ; 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33931944

RESUMO

Targeting autophagy is a promising therapeutic approach in cancer therapy. Here, we screened 30 traditional herbal medicines to identify novel autophagy regulators and found that Platycodon grandiflorus (PG) and platycodin D (PD), a triterpenoid saponin from PG, inhibited autophagy in glioblastoma multiforme (GBM) cells. Mechanistically, PD prevented lysosomal degradation and the fusion between autophagosomes and lysosomes by inducing sequestration of free cholesterol in lysosomes. The autophagy inhibitory effect of PD was mimicked by both genetic and pharmacological inhibition of Niemann-Pick C1 (NPC1), which exports low-density lipoprotein (LDL)-derived cholesterol from lysosomes. Moreover, PD promoted the uptake of exogenous LDL cholesterol via upregulation of LDL receptor (LDLR), leading to further accumulation of cholesterol within lysosomes and GBM cell death. Importantly, these phenomena were more pronounced in LDLR-overexpressing GBM cells than in normal astrocytes. Finally, blockade of cholesterol uptake by LDLR knockdown reversed the PD-induced inhibition of autophagy and GBM cell growth. Our study proposes that PD could be a potent anti-GBM drug by disrupting cholesterol trafficking and autophagy.

4.
Exp Mol Med ; 53(5): 956-972, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34035463

RESUMO

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Saponinas/farmacologia , Serina Endopeptidases/metabolismo , Triterpenos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Antivirais/química , COVID-19/metabolismo , Linhagem Celular , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Modelos Moleculares , Platycodon/química , SARS-CoV-2/fisiologia , Saponinas/química , Triterpenos/química
5.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34050020

RESUMO

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

6.
Am J Chin Med ; 49(3): 767-784, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33657989

RESUMO

Gliomas are the mostly observed form of primary brain tumor, and glioblastoma multiforme (GBM) shows the highest incidence. The survival rate of GBM is fairly poor; thus, discovery of effective treatment options is required. Among several suggested targets for therapy, the Axl/IL-6/STAT3 signaling pathway has gained recent interest because of its important role within cancer microenvironment. Quercetin, a plant flavonoid, is well known for its anticancer action. However, the effect of quercetin on Axl has never been reported. Quercetin treatment significantly reduced cell viability in two GBM cell lines of U87MG and U373MG while keeping 85% of normal astrocytes alive. Further western blot assays suggested that quercetin induces apoptosis but does not affect Akt or mitogen-activated protein kinases, factors related to cell proliferation. Quercetin also decreased IL-6 release and phosphorylation of STAT3 in GBM cells. In addition, gene expression, protein expression, and half-life of synthesized Axl protein were all suppressed by quercetin. By applying shRNA for knockdown of Axl, we could confirm that the role of Axl was crucial in the apoptotic effect of quercetin on GBM cells. In conclusion, we suggest quercetin as a potential anticancer agent, which may improve cancer microenvironment of GBM via the Axl/IL-6/STAT3 pathway.


Assuntos
Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quercetina/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Humanos , Fitoterapia , Quercetina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética
7.
Sci Rep ; 10(1): 19834, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199761

RESUMO

The root of Platycodon grandiflorum (PG) has long been used as a traditional herbal medicine in Asian country. Platycondin D (PD), triterpenoid saponin that is a main constituent of PG, exhibits various biological activities such as anti-inflammatory, anti-oxidant, anti-diabetic, and anti-cancer effects. A previous study showed that PD had cholesterol-lowering effects in mice that develop hypercholesterolemia, but the underlying molecular mechanisms have not been elucidated during the last decade. Here, we demonstrated that both PG and PD markedly increased levels of cell surface low-density lipoprotein receptor (LDLR) by down-regulation of the E3 ubiquitin ligase named inducible degrader of the LDLR (IDOL) mRNA, leading to the enhanced uptake of LDL-derived cholesterol (LDL-C) in hepatic cells. Furthermore, cycloheximide chase analysis and in vivo ubiquitination assay revealed that PD increased the half-life of LDLR protein by reducing IDOL-mediated LDLR ubiquitination. Finally, we demonstrated that treatment of HepG2 cells with simvastatin in combination with PG and PD had synergistic effects on the improvement of LDLR expression and LDL-C uptake. Together, these results provide the first molecular evidence for anti-hypercholesterolemic activity of PD and suggest that PD alone or together with statin could be a potential therapeutic option in the treatment of atherosclerotic cardiovascular disease.


Assuntos
LDL-Colesterol/metabolismo , Hepatócitos/metabolismo , Platycodon/química , Receptores de LDL/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Ubiquitina-Proteína Ligases/genética , Linhagem Celular , Cicloeximida/farmacologia , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Raízes de Plantas/química , Sinvastatina/farmacologia , Ubiquitinação
8.
Exp Neurobiol ; 29(2): 107-119, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32156101

RESUMO

The severe acute respiratory coronavirus 2 (SARS-CoV-2), which emerged in December 2019 in Wuhan, China, has spread rapidly to over a dozen countries. Especially, the spike of case numbers in South Korea sparks pandemic worries. This virus is reported to spread mainly through person-to-person contact via respiratory droplets generated by coughing and sneezing, or possibly through surface contaminated by people coughing or sneezing on them. More critically, there have been reports about the possibility of this virus to transmit even before a virus-carrying person to show symptoms. Therefore, a low-cost, easy-access protocol for early detection of this virus is desperately needed. Here, we have established a real-time reverse-transcription PCR (rtPCR)-based assay protocol composed of easy specimen self-collection from a subject via pharyngeal swab, Trizol-based RNA purification, and SYBR Green-based rtPCR. This protocol shows an accuracy and sensitivity limit of 1-10 virus particles as we tested with a known lentivirus. The cost for each sample is estimated to be less than 15 US dollars. Overall time it takes for an entire protocol is estimated to be less than 4 hours. We propose a cost-effective, quick-and-easy method for early detection of SARS-CoV-2 at any conventional Biosafety Level II laboratories that are equipped with a rtPCR machine. Our newly developed protocol should be helpful for a first-hand screening of the asymptomatic virus-carriers for further prevention of transmission and early intervention and treatment for the rapidly propagating virus.

9.
Front Pharmacol ; 10: 1097, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31607928

RESUMO

Atopic dermatitis (AD) is a frequent skin complication that is caused by unknown reasons. KHU-ATO-JIN-D (KAJD) is a new drug aimed at AD composed of a mixture of extracts from six plants known to have anti-inflammatory and antiallergic effects. This study investigated whether KAJD alleviates 2,4-dinitrochlorobenzene (DNCB)-induced AD in BALB/c mice and several immune cell types. We applied KAJD to DNCB-induced AD-like skin lesions in BALB/c mice, phorbol myristate acetate/ionomycin-stimulated human mast cells (HMC-1), and lipopolysaccharide (LPS)-stimulated macrophages and splenocytes. Histological, ELISA, PCR, and Western blot experiments were performed. The application of KAJD significantly attenuated the lesion severity and skin thickness and inhibited the infiltration of inflammatory cells, mast cells, and CD4+ T cells into the sensitized skin of mice. Reduced leukocyte numbers and proinflammatory cytokine and IgE levels were also observed in the sera of KAJD-treated mice. Moreover, in vitro studies demonstrated that KAJD treatment reduced the LPS-induced expression of proinflammatory cytokines and nitric oxide (NO) production in RAW 264.7 cells. The regulation of IL-4 and IL-6 mRNA and MAPK pathways was also detected in agonist-induced isolated splenocytes and HMC-1 cells by the addition of KAJD. Taken together, our results demonstrate that KAJD inhibits the development of DNCB-induced AD in BALB/c mice and in several immune cell types, suggesting that KAJD might be a useful therapeutic drug for the treatment of AD.

10.
Am J Chin Med ; 47(3): 691-705, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30974965

RESUMO

Scutellaria Radix (SR) is an herb traditionally used in Asian countries to treat inflammatory diseases. Recent studies report that SR exhibits anticancer activities in various types of tumors. In this study, we investigated the apoptotic and autophagic effect of SR in non-small cell lung cancer (NSCLC), the leading cause of cancer-associated death. Treatment of SR in two NSCLC cell lines, H358 and H2087 cells resulted in suppressed cell viability. Western blot assays showed increased expressions of Bcl-2-associated X protein (Bax), cleaved-caspase 3 and cleaved-Poly ADP ribose polymerase (PARP), key factors of apoptosis. Co-treatment of SR with a caspase inhibitor Z-VAD led to nullification of the antiproliferative effect, suggesting the role of apoptosis in the action mechanism of SR. Further experiments revealed autophagy was involved in the effect of SR. SR-treated NSCLC cells expressed increased ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I. When chloroquine was co-treated with SR, this ratio was further increased, indicating SR treatment induced autophagy in NSCLC cells. Interestingly, loss of autophagy by 3-Methyladenine (3-MA) co-treatment suppressed SR-induced apoptosis. We then evaluated the relevance of AMP-activated protein kinase (AMPK) in the autophagic/apoptotic process in NSCLC by SR treatment. Immunoblot assays showed increased phosphorylation of AMPK α and P70-S6 kinase in SR-treated H358 and H2087 cells. Under AMPK-inhibited conditions by compound C, SR treatment failed to induce both autophagy and apoptosis. Taken together, this study identifies the positive effect of SR in H358 and H2087 cells by inducing apoptosis via AMPK-dependent autophagy. Thus, our results suggest the potential use of SR as a novel therapeutic strategy for NSCLC patients.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Extratos Vegetais/farmacologia , Scutellaria baicalensis/química , Humanos , Estimulação Química , Células Tumorais Cultivadas
11.
BMC Complement Altern Med ; 18(1): 215, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30005655

RESUMO

BACKGROUND: Jawoongo is an herbal mixture used in traditional medicine to treat skin diseases. This study aimed to investigate whether Jawoongo ameliorates Atopic dermatitis (AD)-like pathology in mice and to understand its underlying cellular mechanisms. METHODS: AD was induced by 2, 4-Dinitrocholrlbenzene (DNCB) in BALB/c mice. Treatment with Jawoongo was assessed to study the effect of Jawoongo on AD in mice. Histological Analysis, blood analysis, RT-PCR, western blot analysis, ELISA assay and cell viability assay were performed to verify the inhibitory effect of Jawoongo on AD in mice. RESULTS: We found that application of Jawoongo in an ointment form on AD-like skin lesions on DNCB-exposed BALB/c mice reduced skin thickness and ameliorated skin infiltration with inflammatory cells, mast cells and CD4+ cells. The ointment also reduced the mRNA levels of IL-2, IL-4, IL-13 and TNF-α in the sensitized skin. Leukocyte counts and the levels of IgE, IL-6, IL-10 and IL-12 were decreased in the blood of the DNCB-treated mice. Furthermore, studies on cultured cells demonstrated that Jawoongo exhibits anti-inflammatory activities, including the suppression of proinflammatory cytokine expression, nitric oxide (NO) production, and inflammation-associated molecule levels in numerous types of agonist-stimulated innate immune cell, including human mast cells (HMC-1), murine macrophage RAW264.7 cells, and splenocytes isolated from mice. CONCLUSION: These findings indicate that Jawoongo alleviates DNCB-induced AD-like symptoms via the modulation of several inflammatory responses, indicating that Jawoongo might be a useful drug for the treatment of AD.


Assuntos
Angelica/química , Anti-Inflamatórios/administração & dosagem , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/imunologia , Dinitroclorobenzeno/toxicidade , Lithospermum/química , Extratos Vegetais/administração & dosagem , Animais , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/genética , Humanos , Imunoglobulina E/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
BMC Complement Altern Med ; 17(1): 186, 2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28359265

RESUMO

BACKGROUND: Allergic diseases including allergic rhinitis, asthma, and atopic dermatitis are increasing worldwide. Common medications used to treat these inflammatory disorders are anti-histamines and corticosteroids, but they have their own limitations such as short duration and severe side effects. Thus, interest in complementary and alternative medicine is continually growing. Here, we investigate the anti-inflammatory mechanisms of Tonggyu-tang (TGT), a traditional Korean medicine that has been used to treat patients with allergic nasal disorders. METHODS: We measured mRNA expressions and production of pro-inflammatory cytokines such as interleukin (IL)-4, IL-6, IL-8 and tumor necrosis factor alpha (TNF-α) by RT-PCR and ELISA assays in HMC-1 (human mast cell line-1) and HaCaT cells, immortalized human keratinocytes. Moreover, we evaluated the effect of TGT on two major inflammation-related pathways, mitogen activated protein kinase (MAPK) and NF-κB signaling pathway in these two cells. RESULTS: Our results revealed that that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in the agonist-treated HMC-1 and HaCaT cells. We also found that TGT suppressed MAPK signaling pathway including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. CONCLUSION: Taken together, our results demonstrate that TGT inhibits expression of pro-inflammatory cytokines by suppressing MAPK and NF-kB pathway in both mast cells and keratinocytes, suggesting the potential use of TGT in treating allergic inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Queratinócitos/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , NF-kappa B/imunologia , Extratos Vegetais/farmacologia , Anti-Inflamatórios/química , Linhagem Celular , Citocinas/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Queratinócitos/imunologia , Mastócitos/imunologia , Medicina Tradicional Coreana , NF-kappa B/genética , Extratos Vegetais/química
13.
FASEB J ; 31(4): 1461-1481, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28073834

RESUMO

Membrane proteins sense extracellular cues and transduce intracellular signaling to coordinate directionality and speed during cellular migration. They are often localized to specific regions, as with lipid rafts or tetraspanin-enriched microdomains; however, the dynamic interactions of tetraspanins with diverse receptors within tetraspanin-enriched microdomains on cellular surfaces remain largely unexplored. Here, we investigated effects of tetraspan(in) TM4SF5 (transmembrane 4 L6 family member 5)-enriched microdomains (T5ERMs) on the directionality of cell migration. Physical association of TM4SF5 with epidermal growth factor receptor (EGFR) and integrin α5 was visualized by live fluorescence cross-correlation spectroscopy and higher-resolution microscopy at the leading edge of migratory cells, presumably forming TM4SF5-enriched microdomains. Whereas TM4SF5 and EGFR colocalized at the migrating leading region more than at the rear, TM4SF5 and integrin α5 colocalized evenly throughout cells. Cholesterol depletion and disruption in TM4SF5 post-translational modifications, including N-glycosylation and palmitoylation, altered TM4SF5 interactions and cellular localization, which led to less cellular migration speed and directionality in 2- or 3-dimensional conditions. TM4SF5 controlled directional cell migration and invasion, and importantly, these TM4SF5 functions were dependent on cholesterol, TM4SF5 post-translational modifications, and EGFR and integrin α5 activity. Altogether, we showed that TM4SF5 dynamically interacted with EGFR and integrin α5 in migratory cells to control directionality and invasion.-Kim, H.-J., Kwon, S., Nam, S. H., Jung, J. W., Kang, M., Ryu, J., Kim, J. E., Cheong, J.-G., Cho, C. Y., Kim, S., Song, D.-G., Kim, Y.-N., Kim, T. Y., Jung, M.-K., Lee, K.-M., Pack, C.-G., Lee, J. W. Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.


Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colesterol/metabolismo , Receptores ErbB/metabolismo , Glicosilação , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Hepatócitos/ultraestrutura , Humanos , Integrina alfa5/metabolismo , Lipoilação , Microdomínios da Membrana/ultraestrutura , Ligação Proteica , Processamento de Proteína Pós-Traducional
15.
Lung Cancer ; 90(1): 22-31, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26190015

RESUMO

OBJECTIVES: The membrane glycoprotein TM4SF5 (transmembrane 4 L6 family member 5), which is similar to the tetraspanins, is highly expressed in different cancers and causes epithelial-mesenchymal transition (EMT). TM4SF5 interacts with other membrane proteins during its pro-tumorigenic roles, presumably at tetraspanin-enriched microdomains (TEMs/TERMs). Here, we explored TM4SF5-mediated resistance against the clinically important EGFR kinase inhibitors, with regards to cooperation with other membrane proteins, particularly the insulin-like growth factor 1 receptor (IGF1R). MATERIALS AND METHODS: Using cancer cells including NSCLC with TM4SF5 overexpression or IGF1R suppression in either normal 2 dimensional (2D), 3D aqueous spheroids, or 3D collagen I gels systems, the sensitivity to tyrosine kinase inhibitors (TKIs) were evaluated. RESULTS AND CONCLUSION: We found that TM4SF5 and IGF1R transcriptionally modulated one another, with each protein promoting the expressions of the other. Expression of TM4SF5 in gefitinib-sensitive HCC827 cells caused resistance to erlotinib and gefitinib, but not to sorafenib [a platelet derived growth factor receptor (PDGFR) inhibitor]; whereas suppression of IGF1R from gefitinib-resistant NCI-H1299 cells caused enhanced sensitization to the inhibitors. Expression of TM4SF5 and IGF1R in the drug-sensitive cells promoted signaling activities of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and S6 kinase (S6K), and resulted in a higher residual EGFR activity, even after EGFR kinase inhibitor treatment. Complex formation between TM4SF5 and IGF1R was observed, and also included EGFR, dependent on TM4SF5 expression. The TM4SF5-mediated drug resistance was further confirmed in an aqueous 3D spheroid system or upon being embedded in 3D extracellular matrix (ECM)-surrounded gel systems. Collectively, these data suggest that anti-TM4SF5 reagents may be combined with the EGFR kinase inhibitors to enhance the efficacy of chemotherapies against NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptores de Somatomedina/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Receptor IGF Tipo 1 , Receptores de Somatomedina/biossíntese , Receptores de Somatomedina/genética , Transdução de Sinais , Esferoides Celulares
16.
Oncotarget ; 6(25): 21655-74, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26091349

RESUMO

The adhesion properties of cells are involved in tumor metastasis. Although KRS at the plasma membrane is shown important for cancer metastasis, additionally to canonical roles of cytosolic KRS in protein translation, how KRS and its downstream effectors promote the metastatic migration remains unexplored. Disseminative behaviors (an earlier metastatic process) of colon cancer cell spheroids embedded in 3D collagen gels were studied with regards to cell adhesion properties, and relevance in KRS(-/+) knocked-down animal and clinical colon cancer tissues. Time-lapse imaging revealed KRS-dependent cell dissemination from the spheroids, whereas KRS-suppressed spheroids remained static due to the absence of outbound movements supported by cell-extracellular matrix (ECM) adhesion. While keeping E-cadherin at the outward disseminative cells, KRS caused integrin-involved intracellular signaling for ERK/c-Jun, paxillin, and cell-ECM adhesion-mediated signaling to modulate traction force for crawling movement. KRS-suppressed spheroids became disseminative following ERK or paxillin re-expression. The KRS-dependent intracellular signaling activities correlated with the invasiveness in clinical colon tumor tissues and in KRS(-/+) knocked-down mice tissues. Collectively, these observations indicate that KRS at the plasma membrane plays new roles in metastatic migration as a signaling inducer, and causes intracellular signaling for cancer dissemination, involving cell-cell and cell-ECM adhesion, during KRS-mediated metastasis.


Assuntos
Colágeno Tipo I/metabolismo , Neoplasias do Colo/enzimologia , Lisina-tRNA Ligase/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Citosol/metabolismo , Matriz Extracelular/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Técnica Indireta de Fluorescência para Anticorpo , Células HCT116 , Humanos , Camundongos , Metástase Neoplásica , Paxilina/metabolismo , Fosforilação , Biossíntese de Proteínas , Transdução de Sinais
17.
Hepatology ; 61(6): 1978-97, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25627085

RESUMO

UNLABELLED: Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Epithelial-mesenchymal transition (EMT) is related to self-renewal capacity and circulating tumor cell (CTC) characteristics for tumor metastasis. Although tumor metastasis is a life-threatening, complicated process that occurs through circulation of tumor cells, mechanistic aspects of self-renewal and circulating capacities have been largely unknown. Hepatic transmembrane 4 L six family member 5 (TM4SF5) promotes EMT for malignant growth and migration, so it was rationalized that TM4SF5, as a hepatocellular carcinoma (HCC) biomarker, might be important for metastatic potential. Here, self-renewal capacity by TM4SF5 was mechanistically explored using hepatocarcinoma cells with or without TM4SF5 expression, and we explored whether they became CTCs using mouse liver-orthotopic model systems. We found that TM4SF5-dependent sphere growth correlated with CD24(-) , aldehyde dehydrogenase (ALDH) activity, as well as a physical association between CD44 and TM4SF5. Interaction between TM4SF5 and CD44 was through their extracellular domains with N-glycosylation modifications. TM4SF5/CD44 interaction activated proto-oncogene tyrosine-protein kinase Src (c-Src)/signal transducer and activator of transcription 3 (STAT3)/Twist-related protein 1 (Twist1)/B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) signaling for spheroid formation, whereas disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts using 200∼5,000 cells per injection, TM4SF5-positive tumors exhibited subpopulations with locally increased CD44 expressions, supporting for tumor cell differentiation. TM4SF5-positive, but not TM4SF5- or CD44-knocked-down, cells were identified circulating in blood 4-6 weeks after orthotopic liver injection using in vivo laser scanning endomicroscopy. Anti-TM4SF5 reagent blocked their metastasis to distal intestinal organs. CONCLUSION: TM4SF5 promotes self-renewal and CTC properties supported by TM4SF5(+) /CD44(+(TM4SF5-bound)) /ALDH(+) /CD24(-) markers during HCC metastasis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas de Membrana/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Esferoides Celulares , Proteína 1 Relacionada a Twist/metabolismo , Quinases da Família src/metabolismo
18.
Mol Cell Biol ; 35(1): 167-81, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25332235

RESUMO

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study ßTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCF(FBXW11) E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF(FBXW11) bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Contendo Repetições de beta-Transducina/fisiologia , Células HEK293 , Humanos , Mutagênese , Mutagênese Sítio-Dirigida , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma , Proteômica , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Ubiquitina/química
19.
PLoS One ; 9(7): e102817, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25033048

RESUMO

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl4-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFß1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.


Assuntos
Movimento Celular/fisiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Proteínas de Membrana/metabolismo , Invasividade Neoplásica/patologia , Tetraspanina 24/metabolismo , Tetraspanina 30/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fenótipo , Tetraspaninas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
20.
Biochim Biophys Acta ; 1843(9): 2037-54, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24861866

RESUMO

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFß1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colágeno Tipo I/farmacologia , Cortactina/metabolismo , Pseudópodes/metabolismo , Fatores de Transcrição/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Actinas/metabolismo , Animais , Neoplasias da Mama/enzimologia , Bovinos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Cortactina/genética , Feminino , Géis , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Invasividade Neoplásica , Fosfosserina/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-jun/metabolismo , Pseudópodes/efeitos dos fármacos , Transdução de Sinais , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail , Transcrição Genética , Fator de Crescimento Transformador beta1/metabolismo
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