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1.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638566

RESUMO

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.


Assuntos
Células Dendríticas/imunologia , Interleucina-15/biossíntese , Células Matadoras Naturais/imunologia , Receptores de Interleucina-15/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Células Dendríticas/efeitos dos fármacos , Eletroporação , Humanos , Quinase I-kappa B/biossíntese , Quinase I-kappa B/genética , Imunoterapia , Interleucina-15/química , Interleucina-15/genética , Células Matadoras Naturais/efeitos dos fármacos , Leucócitos Mononucleares , NF-kappa B/farmacologia , Cultura Primária de Células , Receptores de Interleucina-15/química , Receptores de Interleucina-15/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
2.
Acta Histochem ; 123(1): 151653, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33246224

RESUMO

OBJECTIVE: Cardiac hypertrophy is an adaptive response to physiological and pathological stimuli, the latter of which frequently progresses to valvulopathy, heart failure and sudden death. Recent reports revealed that pyroptosis is involved in regulating multiple cardiovascular diseases progression, including cardiac hypertrophy. However, the underlying mechanisms remain poorly understood. This study aims to extensively investigate the regulation of miR-133a-3p on pyroptosis in angiotensin II (Ang II)-induced cardiac hypertrophyin vitro. METHODS: The in vitro model of cardiac hypertrophy was induced by Ang II, which was validated by qPCR combined with measurement of cell surface area by immunofluorescence assay. CCK-8 assay and Hochest33342/PI staining was performed to assess pyroptosis. Dual luciferase reporter system was used to verify the direct interaction between miR-133a-3p and IKKε. The effects of miR-133a-3p/IKKε on pyroptosis activation and cardiac hypertrophy markers (Caspase-1, NLRP3, IL-1ß, IL-18, GSDMD, ASC, ANP, BNP and ß-MHC) were evaluated by western blot, ELISA and qPCR. RESULTS: Ang II treatment could induce cardiomyocyte hypertrophy and pyroptosis. The expression of miR-133a-3p was repressed in Ang II-treated HCM cells, and its overexpression could attenuate both pyroptosis and cardiac hypertrophyin vitro. Additionally, IKKε expression was significantly up-regulated in Ang II-induced HCM cells. Dual luciferase reporter system and qPCR validated that miR-133a-3p directly targeted the 3'-UTR of IKKε and suppressed its expression. Moreover, IKKε overexpression impaired the protective function of miR-133a-3p in cardiomyocyte hypertrophy. CONCLUSION: Collectively, miR-133a-3p attenuates Ang II induced cardiomyocyte hypertrophy via inhibition of pyroptosis by targeting IKKε. Therefore, miR-133a-3p up-regulation may be a promising strategy for cardiac hypertrophy treatment.


Assuntos
Cardiomegalia/metabolismo , Regulação Enzimológica da Expressão Gênica , Quinase I-kappa B/biossíntese , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Piroptose , Linhagem Celular , Citocinas/metabolismo , Ativação Enzimática , Humanos , Miócitos Cardíacos/patologia
3.
Int J Oral Sci ; 12(1): 1, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31900382

RESUMO

Odontogenic tumours are a heterogeneous group of lesions that develop in the oral cavity region and are characterized by the formation of tumoural structures that differentiate as teeth. Due to the diversity of their histopathological characteristics and clinical behaviour, the classification of these tumours is still under debate. Alterations in morphogenesis pathways such as the Hedgehog, MAPK and WNT/ß-catenin pathways are implicated in the formation of odontogenic lesions, but the molecular bases of many of these lesions are still unknown. In this study, we used genetically modified mice to study the role of IKKß (a fundamental regulator of NF-κB activity and many other proteins) in oral epithelial cells and odontogenic tissues. Transgenic mice overexpressing IKKß in oral epithelial cells show a significant increase in immune cells in both the oral epithelia and oral submucosa. They also show changes in the expression of several proteins and miRNAs that are important for cancer development. Interestingly, we found that overactivity of IKKß in oral epithelia and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins (p53, or p16 and p19), leads to the appearance of odontogenic tumours that can be classified as ameloblastic odontomas, sometimes accompanied by foci of secondary ameloblastic carcinomas. These tumours show NF-κB activation and increased ß-catenin activity. These findings may help to elucidate the molecular determinants of odontogenic tumourigenesis and the role of IKKß in the homoeostasis and tumoural transformation of oral and odontogenic epithelia.


Assuntos
Células Epiteliais/metabolismo , Genes Supressores de Tumor , Quinase I-kappa B/biossíntese , Mucosa Bucal/patologia , Tumores Odontogênicos/patologia , Odontoma/patologia , RNA Mensageiro/genética , Animais , Western Blotting , Células Epiteliais/patologia , Citometria de Fluxo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Transgênicos , Mucosa Bucal/metabolismo , Tumores Odontogênicos/metabolismo , Odontoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
4.
Spine (Phila Pa 1976) ; 44(12): E707-E714, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31150368

RESUMO

STUDY DESIGN: The effect of triptolide on spinal cord injury (SCI) and inflammatory response was observed by establishing SCI rat model. And in vitro experiments were conducted to determine the underlying mechanism of triptolide-mediated in murine microglial cell line BV2. OBJECTIVE: To determine the underlying mechanism of triptolide in suppressing the microglia activation to improve SCI. SUMMARY OF BACKGROUND DATA: Triptolide, as a major active ingredient of Chinese herb Tripterygium wilfordii, can promote spinal cord repair through inhibiting microglia activation, but the underlying mechanism is not clear. METHODS: Locomotion recovery was accessed by Basso, Beattie, and Bresnahan score, the number of footfalls, stride length, and angle of rotation analysis. Expressions of microRNA 96 (miR-96), microglia activation marker Iba-1, and IκB kinase (IKKß)/nuclear factor (NF)-κB-related proteins were detected by qRT-PCR or western blot. Inflammatory cytokines tumor necrosis factor-α and interleukin -1ß were measured by enzyme-linked immuno sorbent assay. The regulation of miR-96 on IKKß was confirmed by dual luciferase reporter assay. RESULTS: Triptolide promoted locomotion recovery of SCI rats, upregulated the expression of miR-96, decreased microglia activation marker Iba-1 and IKKß/NF-κB-related proteins, and inhibited inflammatory cytokines tumor necrosis factor-α and interleukin-1ß levels in spinal cord tissues and lipopolysaccharide -induced microglia. Triptolide suppressed the microglia activation and inflammatory cytokines secretion in BV2 cells through up-regulating miR-96. We confirmed the interaction between miR-96 and IKKß, and IKKß expression was negatively regulated by miR-96. Finally, we determined that triptolide suppressed the microglia activation and inflammatory cytokines secretion through miR-96/IKKß pathway. CONCLUSION: Triptolide suppressed microglia activation after SCI through miR-96/IKKß/NF-κB pathway. LEVEL OF EVIDENCE: N/A.


Assuntos
Diterpenos/uso terapêutico , Quinase I-kappa B/biossíntese , MicroRNAs/biossíntese , Microglia/metabolismo , NF-kappa B/biossíntese , Fenantrenos/uso terapêutico , Traumatismos da Medula Espinal/metabolismo , Animais , Diterpenos/farmacologia , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Quinase I-kappa B/antagonistas & inibidores , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Lipopolissacarídeos/toxicidade , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Fenantrenos/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Traumatismos da Medula Espinal/tratamento farmacológico
5.
Obesity (Silver Spring) ; 27(5): 767-776, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30933434

RESUMO

OBJECTIVE: Overexpression of inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε) contributes to blunted catecholamine-induced lipolysis. Tumor necrosis factor α (TNF-α) upregulates adipose IKKε expression to inhibit stimulated lipolysis, which can be reversed by IKKε inhibitors. This study investigated adipose IKKε expression in children with and without obesity and potential involvement of the Lin28B/let-7a axis in posttranscriptional regulation of TNF-α-stimulated IKKε in adipocytes. METHODS: Adipose IKKε was detected in children both with and without obesity. The effects of TNF-α on IKKε expression of adipocytes were investigated. Inhibitor and mimics of microRNA let-7a or short interfering RNA of protein lin-28 homolog B (Lin28B) were used to determine the effect of the Lin28B/let-7a axis on TNF-α-mediated IKKε upregulation. Reporter assays were performed to confirm that let-7a targets the IKKε gene. RESULTS: Adipose IKKε expression in children with obesity was upregulated to a greater extent than that in children without obesity and was positively correlated with BMI. TNF-α increased IKKε expression through activation of Lin28B/let-7a and then inhibited isoproterenol-stimulated lipolysis in adipocytes. Blocking the Lin28B /let-7a axis rescued inhibition of isoproterenol-stimulated lipolysis produced by TNF-α by inhibiting IKKε expression. Reporter assays confirmed that IKKε is a target of let-7a. CONCLUSIONS: Adipose IKKε expression in children with obesity is substantially elevated and positively correlated with BMI. TNF-α induces catecholamine resistance via activation of the Lin28B/let-7a/IKKε pathway.


Assuntos
Adipócitos/metabolismo , Catecolaminas/metabolismo , Quinase I-kappa B/biossíntese , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3-L1 , Animais , Criança , Feminino , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B/genética , Masculino , Camundongos , MicroRNAs/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Transfecção , Regulação para Cima
6.
Dig Dis Sci ; 64(5): 1204-1216, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30560335

RESUMO

BACKGROUND: IκB kinase 2 (IKK2) is the primary catalytic subunit of the IKK complex. Activation of IKK phosphorylates the inhibitors of NF-κB (IκB), triggering the translocation of NF-κB. AIMS: Although IKK2 has been investigated in the inflammation-cancer transformation of gastric epithelium, its role in gastric cancer (GC) cells remained unexplored. METHODS: The IKK2 distribution and expression were measured by immunochemistry staining in clinical specimens. The proliferation, apoptosis, and migration of GC cells were analyzed after IKK2 expression intervention. Using Erk and ß-catenin inhibitors, we investigated the relationship between IKK2 and Erk and ß-catenin pathways. In the GC-burdened mice, we confirmed the effects of IKK2 inhibition on tumor growth. RESULTS: Here, we found that IKK2 expression in the GC area was even higher than adjacent inflammatory area, and the GC patients with high expression of IKK2 showed worse overall and disease-free survival. Introduction of IKK2 inhibitor SC-514 inhibited the cell proliferation and induced apoptosis of SGC-7901 cells, in turn overexpression of IKK2 in MGC-823 cells showed the reverse effects. The proliferative activity of IKK2 on GC cells was dependent on the activation of ß-catenin and Erk pathways. Additionally, IKK2 alteration affected the migration of GC cells. In vivo, IKK2 inhibition mitigated the tumor growth. Decreased expression of PCNA as well as an increase in cleaved caspase 3 and p53 were observed. CONCLUSION: Our results indicate that IKK2 promotes the GC cell proliferation and inhibits their apoptosis, suggesting it may be a potential target for GC therapy.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/biossíntese , Neoplasias Gástricas/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Distribuição Aleatória , Neoplasias Gástricas/patologia , Tiofenos/farmacologia
7.
Clin Transl Oncol ; 20(12): 1592-1603, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29796997

RESUMO

PURPOSE: Uveal melanoma, although a rare form of cancer, is the most common primary malignancy of the eye in adults. Nuclear factor-κB (NF-κB) is a transcription factor that transactivates genes involved in the regulation of cell growth, apoptosis, angiogenesis, and metastasis, but the molecular mechanisms that negatively regulate NF-κB activation are not fully understood. NF-κB can also be activated by DNA damage pathway through NEMO protein. Therefore, the objective of this study is to elucidate the role of NEMO/IKKγ protein in uveal melanoma patients. METHODS: Seventy-five formalin-fixed paraffin-embedded prospective tissues of uveal melanoma were included in the present study. These cases were reviewed and investigated for the expression of NEMO/IKKγ protein by immunohistochemistry and validated by western blotting along with the qRT-PCR for mRNA expression. Expression levels were correlated with the clinicopathological parameters and patients' outcome. RESULTS: Immunohistochemistry showed cytoplasmic expression of NEMO/IKKγ expression in only 22 out of 75 (29.33%) cases. This result was confirmed by western blotting, and correlated well with the immunohistochemical expression of NEMO/IKKγ protein (48 kDa). In addition, downregulation of this gene was found in 87.93% of the cases when compared with the normal tissues. On statistical analysis, loss of NEMO/IKKγ protein was correlated with neovascularization, high mitotic count, and presence of vascular loop (p < 0.05). There was less overall survival rate with low expression of NEMO/IKKγ protein in patients with uveal melanoma. CONCLUSION: This was the first study suggesting the relevant role of NEMO/IKKγ protein, and highlights the prognostic significance with outcome in uveal melanoma patients. This protein might be used as a screening biomarker in these patients after large-scale validation and translational studies.


Assuntos
Biomarcadores Tumorais/análise , Quinase I-kappa B/biossíntese , Melanoma/patologia , Neoplasias Uveais/patologia , Adulto , Idoso , Feminino , Humanos , Quinase I-kappa B/análise , Masculino , Melanoma/metabolismo , Melanoma/mortalidade , Pessoa de Meia-Idade , Prognóstico , Neoplasias Uveais/metabolismo , Neoplasias Uveais/mortalidade
8.
Inflamm Res ; 67(5): 455-466, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29523916

RESUMO

OBJECTIVE: The peptide lycosin-I has anti-bacterial and anti-cancer capacities. However, the anti-inflammatory activity of lycosin-I remains unknown. We investigated whether lycosin-I could attenuate inflammation. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with lycosin-I before exposure to tumor necrosis factor-α (TNF-α). The expression of intercellular cell adhesion molecule-1 (ICAM-1), nuclear transcription factor-kappa B (NF-κB) p65 and inhibitory subunit of NF-κB alpha (IκBα) was evaluated by western blot. The expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) was detected by quantitative RT-PCR or ELISA. Immunofluorescence analysis was used to determine the impact of lycosin-I on NF-κB pathway. C57BL/6 mice were pretreated with lycosin-I before exposure with lipopolysaccharide (LPS). RESULTS: Lycosin-I significantly reduced the TNF-α-enhanced expression of IL-6, IL-8 and ICAM-1. Lycosin-I also inhibited the human monocyte cells adhesion to HUVECs. We further demonstrated that lycosin-I could effectively suppress the reaction of endothelial cells to TNF-α by inhibiting IκBα degradation. Subsequently, the phosphorylation and translocation of NF-κB p65 could also be attenuated. Furthermore, lycosin-I exhibited a significant protection of C57BL/6 mice against LPS-induced death. CONCLUSIONS: Our results suggested that the anti-inflammatory activity of lycosin-I was associated with NF-κB activation and lycosin-I had potential to be a novel therapeutic candidate for inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Inflamação/prevenção & controle , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Venenos de Aranha/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Quinase I-kappa B/biossíntese , Inflamação/induzido quimicamente , Inflamação/mortalidade , Molécula 1 de Adesão Intercelular/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/biossíntese , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/biossíntese
9.
Mol Reprod Dev ; 85(1): 46-61, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29219221

RESUMO

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.


Assuntos
Antioxidantes/farmacologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Licopeno/farmacologia , Oócitos/crescimento & desenvolvimento , Acil-CoA Desidrogenase de Cadeia Longa/biossíntese , Animais , Blastocisto/citologia , Proteína Morfogenética Óssea 15/biossíntese , Caspase 3/análise , Caspase 9/análise , Bovinos , Coenzima A Ligases/biossíntese , Fator 9 de Diferenciação de Crescimento/biossíntese , Quinase I-kappa B/biossíntese , NADH Desidrogenase/biossíntese , Superóxido Dismutase/biossíntese
10.
Mol Biol (Mosk) ; 51(3): 447-459, 2017.
Artigo em Russo | MEDLINE | ID: mdl-28707661

RESUMO

In murine bone-marrow stromal microenvironment cells and in human multipotent mesenchymal stromal cells (MMSCs), proinflammatory cytokine interleukin-1 beta (IL-1ß) serves as a growth factor. In murine bone tissue, IL-1ß expression increases in vivo after irradiation. Here, we have presented our evaluation of the effects of exogenous IL-1ß on the expression of NF-kB transcription factors in human MMSCs and stromal layer cells of murine long-term bone marrow cultures (LTBMCs). The cytokine signaling pathway was also activated in murine LTBMC by braking electron radiation in doses of 3-12 Gy. The level of expression of genes that code for IL-1ß, IL-1ß type-I receptor and NF-kB and IKK protein families have been studied at different time points post exposure. In both human and murine stromal cells, exogenous IL-1ß led to an increase in the level of expression of its own gene, while levels of expression of NF-kB and IKK gene families were not substantially changed. Nevertheless, in human cells, a significant correlation between levels of expression of IL-1ß and all NF-kB family genes was detected. It points to a similarity in IL-1ß signal pathways in mesenchymal and hematopoietic cells, where the posttranslational modifications of NF-kB transcription factors play a major role. The irradiation of murine LTBMC resulted in a transient increase in the expression of genes that code NF-kB transcription factors and IL-1ß. These results indicate an important role of Rel, Rela, Relb, and Nfkb2 genes in the induction of IL-1ß signal pathway in murine stromal cells. An increase in IL-1ß expression after the irradiation of stromal cells may be related to both the induction of inflammation due to massive cell death and to a profound stimulation of the expression of this proinflammatory cytokine expression.


Assuntos
Interleucina-1beta/biossíntese , Células-Tronco Mesenquimais/metabolismo , Subunidade p52 de NF-kappa B/genética , Fator de Transcrição RelA/genética , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos da radiação , Células Cultivadas , Raios gama , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Quinase I-kappa B/biossíntese , Inflamação/genética , Inflamação/patologia , Interleucina-1beta/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos da radiação , Camundongos , NF-kappa B/biossíntese , Subunidade p52 de NF-kappa B/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fator de Transcrição RelA/biossíntese
11.
Mol Cancer Res ; 15(9): 1255-1264, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28584022

RESUMO

IKKß (encoded by IKBKB) is a protein kinase that regulates the activity of numerous proteins important in several signaling pathways, such as the NF-κB pathway. IKKß exerts a protumorigenic role in several animal models of lung, hepatic, intestinal, and oral cancer. In addition, genomic and proteomic studies of human tumors also indicate that IKBKB gene is amplified or overexpressed in multiple tumor types. Here, the relevance of IKKß in skin cancer was determined by performing carcinogenesis studies in animal models overexpressing IKKß in the basal skin layer. IKKß overexpression resulted in a striking resistance to skin cancer development and an increased expression of several tumor suppressor proteins, such as p53, p16, and p19. Mechanistically, this skin tumor-protective role of IKKß is independent of p53, but dependent on the activity of the Ink4a/Arf locus. Interestingly, in the absence of p16 and p19, IKKß-increased expression favors the appearance of cutaneous spindle cell-like squamous cell carcinomas, which are highly aggressive tumors. These results reveal that IKKß activity prevents skin tumor development, and shed light on the complex nature of IKKß effects on cancer progression, as IKKß can both promote and prevent carcinogenesis depending on the cell type or molecular context.Implications: The ability of IKKß to promote or prevent carcinogenesis suggests the need for further evaluation when targeting this protein. Mol Cancer Res; 15(9); 1255-64. ©2017 AACR.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinase I-kappa B/biossíntese , Neoplasias Cutâneas/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Epigênese Genética , Quinase I-kappa B/genética , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
12.
Oncotarget ; 8(10): 16233-16242, 2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-27145266

RESUMO

Inhibitor of kappa B kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IKKs. IKKε and TBK1 share the kinase domain and are similar in their ability to activate the nuclear factor-kappa B signaling pathway. IKKε and TBK1 are overexpressed through multiple mechanisms in various human cancers. However, the expression of IKKε and TBK1 in gastric cancer and their role in prognosis have not been studied.To investigate overexpression of the IKKε and TBK1 proteins in gastric cancer and their relationship with clinicopathologic factors, we performed immunohistochemical staining using a tissue microarray. Tissue microarray samples were obtained from 1,107 gastric cancer patients who underwent R0 gastrectomy with extensive lymph node dissection and adjuvant chemotherapy.We identified expression of IKKε in 150 (13.6%) and TBK1 in 38 (3.4%) gastric cancers. Furthermore, co-expression of IKKε and TBK1 was identified in 1.5% of cases. Co-expression of IKKε and TBK1 was associated with differentiated intestinal histology and earlier T stage. In a multivariate binary logistic regression model, intestinal histologic type by Lauren classification and early AJCC stage were significant predictors for expression of IKKε and TBK1 proteins in gastric cancer. Changes in IKKε and TBK1 expression may be involved in the development of intestinal-type gastric cancer. The overexpression of IKKε and TBK1 should be considered in selected patients with intestinal-type gastric cancer.In conclusion, this is the first large-scale study investigating the relationships between expression of IKKε and TBK1 and clinicopathologic features of gastric cancer. The role of IKKε and TBK1 in intestinal-type gastric cancer pathogenesis should be elucidated by further investigation.


Assuntos
Quinase I-kappa B/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Neoplasias Gástricas/metabolismo , Estômago/enzimologia , Feminino , Humanos , Imuno-Histoquímica , Intestinos/patologia , Estimativa de Kaplan-Meier , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Estudos Retrospectivos , Estômago/patologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/terapia , Análise Serial de Tecidos
13.
Cancer Res ; 76(24): 7254-7264, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27923829

RESUMO

Aberrant expression of the kinase IKKε in pancreatic ductal adenocarcinoma (PDAC) has been associated with poor prognosis. In this study, we define a pathobiologic function for IKKε in reprogramming glucose metabolism and driving progression in PDAC. Silencing IKKε in PDAC cells, which overexpressed it endogenously, was sufficient to reduce malignant cell growth, clonogenic potential, glucose consumption, lactate secretion, and expression of genes involved in glucose metabolism, without impacting the basal oxygen consumption rate. IKKε silencing also attenuated c-Myc in a manner associated with diminished signaling through an AKT/GSK3ß/c-MYC phosphorylation cascade that promoted MYC nuclear accumulation. In an orthotopic mouse model, IKKε-silenced PDAC exhibited a relative reduction in glucose uptake, tumorigenicity, and metastasis. Overall, our findings offer a preclinical mechanistic rationale to target IKKε to improve the therapeutic management of PDAC in patients. Cancer Res; 76(24); 7254-64. ©2016 AACR.


Assuntos
Carcinoma Ductal Pancreático/patologia , Glucose/metabolismo , Quinase I-kappa B/biossíntese , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Neoplasias Pancreáticas/metabolismo , Reação em Cadeia da Polimerase , Regulação para Cima
14.
Oncol Rep ; 36(5): 2771-2776, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27667548

RESUMO

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, apoptosis and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified baicalein from Scutellaria baicalensis as an inhibitor of NF-κB activation. As examined by the NF-κB luciferase reporter assay, we found that baicalein suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. It also inhibited TNF-α-induced nuclear translocation of p65 through inhibition of phosphorylation and degradation of IκBα. Furthermore, baicalein blocked the TNF-α-induced expression of NF-κB target genes involved in anti-apoptosis (cIAP-1, cIAP-2, FLIP and BCL-2), proliferation (COX-2, cyclin D1 and c-Myc), invasion (MMP­9), angiogenesis (VEGF) and major inflammatory cytokines (IL-8 and MCP1). The flow cytometric analysis indicated that baicalein potentiated TNF-α-induced apoptosis and induced G1 phase arrest in HeLa cells. Moreover, baicalein significantly blocked activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2). Our results imply that baicalein could be a lead compound for the modulation of inflammatory diseases as well as certain cancers in which inhibition of NF-κB activity may be desirable.


Assuntos
Flavanonas/administração & dosagem , Extratos Vegetais/administração & dosagem , Fator de Transcrição RelA/biossíntese , Fator de Necrose Tumoral alfa/genética , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Quinase I-kappa B/biossíntese , Quinase I-kappa B/genética , NF-kappa B/biossíntese , NF-kappa B/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosforilação , Scutellaria baicalensis , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/genética
15.
Br J Cancer ; 115(6): 731-40, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27537390

RESUMO

BACKGROUND: Dysregulation of microRNAs (miRNAs) has been found in human epithelial ovarian cancer (EOC). However, the role and mechanism of action of miR-23a in EOC remain unclear. METHODS: The roles of miR-23a, IKKα, and ST7L in EOC were determined by MTT, colony formation, wounding healing, transwell, flow cytometry, immunofluorescence, RT-qPCR, and western blotting experiments. miR-23a target genes were validated by EGFP reporter assays, RT-qPCR, and western blotting analysis. RESULTS: miR-23a is upregulated and promotes tumorigenic activity by facilitating the progress of cell cycle and EMT and repressing apoptosis in EOC cells. miR-23a enhances the expression of IKKα but suppresses the expression of ST7L by binding the 3'UTR of each transcript in EOC cells. The proliferation, migration, and invasion of EOC cells are increased by IKKα and inhibited by ST7L. Furthermore, miR-23a activates NF-κB by upregulating IKKα and WNT/MAPK pathway by downregulating ST7L. CONCLUSIONS: miR-23a functions as an oncogene by targeting IKKα and ST7L, thus contributing to the malignancy of EOC cells.


Assuntos
Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Quinase I-kappa B/fisiologia , MicroRNAs/genética , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/fisiologia , Regiões 3' não Traduzidas/genética , Carcinoma/patologia , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Feminino , Genes Reporter , Vetores Genéticos , Humanos , Quinase I-kappa B/biossíntese , Quinase I-kappa B/genética , Sistema de Sinalização das MAP Quinases/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Proteínas Supressoras de Tumor , Via de Sinalização Wnt/genética
16.
FEBS J ; 283(12): 2259-71, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26433127

RESUMO

MicroRNAs (miRNAs) act as important post-transcriptional regulators of gene expression in diverse signalling pathways. However, the relationship between miR-200b and the nuclear factor-κB (NF-κB) signalling pathway remains poorly understood in breast cancer cells. In the current study, we show that IKBKB is a direct target of miR-200b, and that miR-200b downregulates IKBKB expression via directly binding to its 3'-UTR. miR-200b inhibits IκBα phosphorylation, nuclear p50/p65 expression, NF-κB-binding activity, and the translocation of p65 to the nucleus. In addition, miR-200b also suppresses tumour necrosis factor (TNF)-α-induced NF-κB activation and the expression of NF-κB target genes. Importantly, IKBKB overexpression attenuates the inhibitory roles of miR-200b in NF-κB expression, NF-κB-binding activity, and the nuclear translocation of p65. We also show that NF-κB p65 knockdown reduces the binding of NF-κB to the miR-200b promoter and miR-200b promoter activity. Furthermore, p65 knockdown or inhibition of IκBα phosphorylation suppresses miR-200b expression. Finally, functional studies show that IKBKB overexpression can restore the cell growth and migration that are suppressed by miR-200b. In conclusion, our results demonstrate that miR-200b, a transcriptional target of NF-κB, suppresses breast cancer cell growth and migration, and NF-κB activation, through downregulation of IKBKB, indicating that miR-200b has potential as a therapeutic target in breast cancer patients.


Assuntos
Neoplasias da Mama/genética , Quinase I-kappa B/genética , MicroRNAs/genética , NF-kappa B/genética , Fator de Transcrição RelA/genética , Apoptose/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Quinase I-kappa B/biossíntese , MicroRNAs/biossíntese , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais
17.
J Mol Med (Berl) ; 94(1): 71-82, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26296700

RESUMO

UNLABELLED: TNF is highly expressed in synovial tissue of rheumatoid arthritis (RA) patients, where it induces proinflammatory cytokine secretion. However, in other cases, TNF will cause cell death. Considering the abnormal proliferation and activation of rheumatoid arthritis synovioblasts, the proper rate of synovioblast apoptosis could possibly relieve arthritis. However, the mechanism mediating TNF-induced synovioblast survival versus cell death in RA is not fully understood. Our objective was to study the role of CD147 in TNF downstream pathway preference in RA synovioblasts. We found that overexpressing TNF in synovial tissue did not increase the apoptotic level and, in vitro, TNF-induced mild synovioblast apoptosis and promoted IL-6 secretion. CD147, which was highly expressed in rheumatoid arthritis synovial fibroblasts (RASFs), increased the resistance of synovioblasts to apoptosis under TNF stimulation. Downregulating CD147 both increased the apoptotic rate and inhibited IκB kinase (IKK)/IκB/NF-κB pathway-dependent proinflammatory cytokine secretion. Further, we determined that it was the extracellular portion of CD147 and not the intracellular portion that was responsible for synovioblast apoptosis resistance. CD147 monoclonal antibody inhibited TNF-induced proinflammatory cytokine production but had no effect on apoptotic rates. Thus, our study indicates that CD147 is resistant to TNF-induced apoptosis by promoting IKK/IκB/NF-κB pathway, and the extracellular portion of CD147 is the functional region. KEY MESSAGES: CD147 inhibits TNF-stimulated RASF apoptosis. CD147 knockdown decreases IKK expression and inhibits NF-κB-related cytokine secretion. CD147's extracellular portion is responsible for apoptosis resistance. CD147 antibody inhibits TNF-related cytokine secretion without additional apoptosis.


Assuntos
Apoptose/fisiologia , Artrite Reumatoide/patologia , Basigina/metabolismo , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Basigina/genética , Células Cultivadas , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Quinase I-kappa B/biossíntese , Proteínas I-kappa B/biossíntese , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Interferência de RNA , RNA Interferente Pequeno , Membrana Sinovial/citologia
18.
Cell Death Differ ; 23(1): 89-98, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26024391

RESUMO

The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-ß, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.


Assuntos
Apoptose/genética , GTP Fosfo-Hidrolases/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular , Fibroblastos/metabolismo , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/biossíntese , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NF-kappa B/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteína X Associada a bcl-2/genética
19.
PLoS One ; 10(8): e0135257, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26301506

RESUMO

Synthetic triterpenoids are antioxidant inflammation modulators (AIMs) that exhibit broad anticancer activity. AIMs bind to KEAP1 and inhibit its ability to promote NRF2 degradation. As a result, NRF2 increases transcription of genes that restore redox balance and reduce inflammation. AIMs inhibit tumor growth and metastasis by increasing NRF2 activity in the tumor microenvironment and by modulating the activity of oncogenic signaling pathways, including NF-κB, in tumor cells. Accumulating evidence suggests that KEAP1 loss or mutation--which results in high levels of sustained NRF2 activity--may promote cancer growth and increase chemoresistance. Loss of KEAP1 also increases the levels of other oncogenic proteins, including IKKß and BCL2. The apparent survival advantage provided to some tumor cells by loss of functional KEAP1 raises the question of whether pharmacological inhibition of KEAP1 could promote tumor growth. To address this issue, we characterized the basal levels of KEAP1 and NRF2 in a panel of human tumor cell lines and profiled the activity of an AIM, RTA 405. We found that in tumor cell lines with low or mutant KEAP1, and in Keap1-/- murine embryonic fibroblasts, multiple KEAP1 targets including NRF2, IKKß, and BCL2 were elevated. Keap1-/- murine embryonic fibroblasts also had higher rates of proliferation and colony formation than their wild-type counterparts. In cells with functional KEAP1, RTA 405 increased NRF2 levels, but not IKKß or BCL2 levels, and did not increase cell proliferation or survival. Moreover, RTA 405 inhibited growth at similar concentrations in cells with different basal NRF2 activity levels and in cells with wild-type or mutant KRAS. Finally, pre-treatment with RTA 405 did not protect tumor cells from doxorubicin- or cisplatin-mediated growth inhibition. Collectively, these data demonstrate that pharmacological activation of NRF2 by AIMs is distinct from genetic activation and does not provide a growth or survival advantage to tumor cells.


Assuntos
Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Ácido Oleanólico/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteína 1 Associada a ECH Semelhante a Kelch , Células MCF-7 , Camundongos , Fator 2 Relacionado a NF-E2/biossíntese , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/síntese química , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transdução de Sinais/efeitos dos fármacos
20.
Biochem Biophys Res Commun ; 464(2): 586-9, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26163261

RESUMO

Inhibitor of kappa B (IκB)-ζ, a member of the nuclear IκB family of proteins, is induced by the transforming growth factor (TGF)-ß signaling pathway and plays a pivotal role in maintaining the balance of T helper (Th) cell subsets. IκB-ζ deficiency results in reduced percentages of Th17 cells and increased percentages of Th1 cells. In this study, the effects of IκB-ζ deficiency on T-cell subsets were examined further. The data showed that IκB-ζ-deficient T cells had a high capacity for generation of regulatory T cells (Tregs) when T cells were cultured under TGF-ß stimulation in the presence of cytokine-neutralizing antibodies. Mechanistically, IκB-ζ itself negatively regulated activation of the Foxp3 promoter in a nuclear factor of kappaB-dependent manner. Thus, this study showed that IκB-ζ controlled Treg differentiation.


Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/fisiologia , Quinase I-kappa B/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Linhagem Celular Tumoral , Humanos , Quinase I-kappa B/biossíntese , Regiões Promotoras Genéticas
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