RESUMO
To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.
Assuntos
Apresentação de Antígeno , Células da Medula Óssea , Células Dendríticas , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Linfócitos T Citotóxicos , Ubiquitina , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitina/metabolismo , Linfócitos T Citotóxicos/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Apresentação de Antígeno/imunologia , Camundongos Endogâmicos C57BL , Fosforilação , Ativação Linfocitária , Diferenciação Celular , Mutação , Morfolinas/farmacologia , Teste de Cultura Mista de Linfócitos , Proliferação de Células , Antígeno B7-2/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Células Cultivadas , Cromonas/farmacologia , Wortmanina/farmacologia , Androstadienos/farmacologiaRESUMO
Matrix metalloproteinases (MMPs) and the epithelial-mesenchymal transition (EMT) contribute to metastasis. As shown in our previous studies, interleukin-6 (IL-6) induces ATM phosphorylation to increase MMP expression and metastasis in lung cancer. However, the exact roles of ATM activation in the IL-6-induced epithelial-mesenchymal transition and lung cancer metastasis are currently unclear. Here, ATM phosphorylation exerts its pro-metastatic effect via vimentin-mediated epithelial-mesenchymal transition, which was supported by the evidence described below. Firstly, IL-6 treatment increases vimentin expression via the ATM-NF-κB pathway. Second, ATM inactivation not only abolishes IL-6-induced increases in vimentin expression but also inhibits IL-6-induced nest formation in a xenograft lung metastasis model. Moreover, close positive correlations were observed between ATM phosphorylation and vimentin upregulation, IL-6 levels and metastasis in lung cancer specimens. Hence, ATM modulates vimentin expression to facilitate IL-6-induced epithelial-mesenchymal transition and metastasis in lung cancer, indicating that ATM and vimentin might be potential therapeutic targets for inflammation-associated lung cancer metastasis.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Interleucina-6/farmacologia , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Vimentina/genética , Células A549 , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Vimentina/metabolismoRESUMO
Although it is known that ataxia-telangiectasia mutated (ATM) and interleukin 6 (IL-6) contribute to multiple drug resistance (MDR) in tumor chemotherapy, the exact role of ATM activation in MDR resulting from increased IL-6 expression is still unclear. In the present study, we demonstrate that the activation of the ATM-NF-kappaB pathway, resulting from increased IL-6 expression, plays a central role in augmented chemoresistance in lung cancer cell lines. This result was supported by the increased expressions of Bcl-2, Mcl-1, Bcl-xl, and the upregulation of MDR-associated protein ABCG2. The higher level of IL-6 reveals not only higher ATM/NF-kappaB activity but also increased expressions of ABCG2, Bcl-2, Mcl-1 and Bcl-xl. Most importantly, lung cancer cells themselves upregulated IL-6 secretion by activating the p38/NF-kappaB pathway through treatment with cisplatin and camptothecin. Taken together, these findings demonstrate that chemotherapeutic agents increase IL-6 expression, hence activating the ATM/NF-kappaB pathway, augmenting anti-apoptotic protein expression and contributing to MDR. This indicates that both IL-6 and ATM are potential targets for the treatment of chemotherapeutic resistance in lung cancer.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Resistencia a Medicamentos Antineoplásicos , Interleucina-6/fisiologia , NF-kappa B/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piridinas/farmacologia , Sulfonas/farmacologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: To explore the role of 4-1BBL in nicotine-treated immature dendritic cells (imDCs) mediated anti-tumor effects. METHODS: Bone marrow-derived imDCs were stimulated with nicotine and 4-1BBL expression was determinated by flow cytometry, Western blot and RT-PCR respectively. Then, the roles of 4-1BBL in nicotine-augmented DCs-dependent T cell proliferation, CTL priming and anti-tumor effects were investigated by BrdU cell proliferation assay, enzyme-linked immunospot assay and in vivo preventive effect on tumor development, respectively. Finally, using relative kinase inhibitors, the mechanism of 4-1BBL up-regulation by nicotine stimulation and the roles of Mek-PI3K signal pathways in nicotine-augmented DCs-dependent T cell proliferation were explored by Western blot and BrdU cell proliferation assay, respectively. RESULTS: Firstly, nicotine could up-regulate 4-1BBL expression in both protein and mRNA levels. Secondly, the effects of nicotine-augmented DCs-dependent T-cell proliferation, CTL priming and anti-tumor effects could be significantly abolished by blocking CD80, CD86 and 4-1BBL activity, respectively. Thirdly, the combined blockages of CD80/CD86, CD80/4-1BBL, CD86/4-1BBL or CD80/CD86/4-1BBL signals could decrease 53.2 %, 29.6 %, 27.9 % and 54.5 % nicotine-enhanced T cell proliferation, respectively. Importantly, nicotine-induced 4-1BBL up-regulation could be decreased by the usage of Mek-PI3K pathway kinase inhibitors. The pre-treatment of Mek-p38-PI3K kinase inhibitors could obviously abolish nicotine-augmented DCs-dependent T cell proliferation. CONCLUSIONS: CD80/CD86 and 4-1BBL are critical for nicotine augmented DCs-mediated anti-tumor effects. 4-1BBL and CD80/CD86 could be considered as potential candidates for preventive and therapeutic tumor vaccination.
Assuntos
Ligante 4-1BB/biossíntese , Transplante de Medula Óssea/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/transplante , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Nicotina/farmacologia , Fosfatidilinositol 3-Quinase/fisiologia , Ligante 4-1BB/genética , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Transplante de Medula Óssea/métodos , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/uso terapêutico , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , MAP Quinase Quinase Quinases/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , Regulação para Cima/imunologiaRESUMO
K48-linked ubiquitination determines antigen degradation and plays vital roles in the process of cross-presentation of bone marrow precursor cell (BMPC)-derived dendritic cells (DCs). Although previous studies revealed that K48 and K27-linked ubiquitination regulates innate immunity, the exact roles of K48 and K27-linked ubiquitination in cross-presentation and BMPC-based adaptive immunity are still uncertain. In this study, we investigated the effects of K48- and K27-mutant ubiquitin (Ub) on BMPC-based adaptive immune response by observing the effects of MG132, Ub deficiency, and K48/K27-mutant Ub on cross-presentation, T cell proliferation, IFN-γ secretion, BMPC-based CTL priming, and thereby the efficiency of cytolytic capacity of BMPC-activate T cells. We demonstrated that MG132, Ub deficiency, and K48-mutant Ub impair cross-presentation, T cell proliferation, IFN-γ secretion, BMPC-based CTL priming, and the cytolytic capacity of BMPC-activated T cells. Moreover, although K27-only Ub decreases cross-presentation, the replenishment of K27-mutant Ub restores Ub deficiency impaireds the abilities of T cell proliferation, IFN-γ secretion, CTL priming, and the cytolytic capacity of BMPC-activated T cells. Thus, these data suggest that K48- and K27-linked ubiquitination contributes to BMPC-mediated adaptive immune response by affecting BMPC cross-presentation and the cytolytic capacity by up-regulating both perforin and granzyme B in BMPC-activated T cells. K48- and K27-mutant Ub might have potential clinical therapeutic function in adaptive immune response-associated diseases.
Assuntos
Apresentação Cruzada , Ubiquitina , Imunidade Adaptativa/genética , Medula Óssea/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
INTRODUCTION: Our previous studies have revealed that nicotine-treated immature dendritic cells (imDCs) have anti-tumor effects in murine lymphoma models. The present study is to explore the preventive and therapeutic anti-tumor effects of nicotine-treated imDCs in murine lung and liver cancer. MATERIALS AND METHODS: To address this objection, bone marrow-derived imDCs were firstly stimulated by nicotine in vitro and the expressions of CD80, CD86, CD40, CD11b, MHC class I and II were determined by flow cytometry. Then, DCs-dependent tumor-lysate-specific T cell proliferation, IL-12(p40+p70) secretion were determined by BrdU cell proliferation assay and enzyme-linked immunosorbent assay, respectively. The anti-tumor effects of such imDCs were further explored by intraperitoneal transfer against tumor challenge or implantation. By using kinase inhibitors, the mechanism of nicotine upregulating CD80 was finally explored by flow cytometry. RESULTS: The results showed that: firstly, nicotine could upregulate the expressions of CD80, CD86, CD40,CD11b, MHC class I and II molecules in imDCs. Secondly, nicotine could promote imDCs-dependent T cell priming and IL-12 secretion. Most importantly, systemic transfer of ex vivo nicotine-stimulated imDCs, which enhanced CD80 expression through PI3K activation, could reveal preventive and effectively therapeutic effects on tumor development. CONCLUSIONS: Ex vivo nicotine stimulation can significantly improve imDCs efficacy for adaptive therapy of cancer. Nicotine-treated imDCs might be considered as a potential candidate for therapeutic tumor immunotherapy for lung and liver cancer.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias Hepáticas , Neoplasias Pulmonares , Nicotina/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/farmacologia , Linfócitos T/imunologia , Regulação para CimaRESUMO
K48-linked ubiquitination determining antigen degradation and the endosomal recruitments of p97 and Sec61 plays vital roles in dendritic cell (DC) cross-presentation. Our previous studies revealed that nicotine treatment increases bone marrow-derived dendritic cell (BM-DC) cross-presentation and promotes BM-DC-based cytotoxic T lymphocyte (CTL) priming. But the effect of nicotine on K48-linked ubiquitination and the mechanism of nicotine-increased BM-DC cross-presentation are still uncertain. In this study, we first demonstrated that ex vivo nicotine administration obviously increased K48-linked ubiquitination in BM-DC. Then, we found that K48-linked ubiquitination was essential for nicotine-augmented cross-presentation, BM-DC-based CTL priming, and thereby the superior cytolytic capacity of DC-activated CTL. Importantly, K48-linked ubiquitination was verified to be necessary for nicotine-augmented endosomal recruitments of p97 and Sec61. Importantly, mannose receptor (MR), which is an important antigenic receptor for cross-presentation, was exactly catalyzed with K48-linked ubiquitination by the treatment with nicotine. Thus, these data suggested that K48-linked ubiquitination contributes to the superior adaptive immunity of nicotine-administrated BM-DC. Regulating K48-linked ubiquitination might have therapeutic potential for DC-mediated immune therapy.
RESUMO
This study tested the hypothesis that PI3K-Akt activity contributes to the superior immune function of IL-15-administrated bone marrow precursor cells (BMPC). Our previous studies revealed that PI3K-Akt play vital role in dendritic cells (DCs) cross-presentation and DC-based CTL priming. Despite the fact that IL-15 serves multiple functions in its therapeutic potential for the induction and maintenance of T cell response, the exact role of PI3K-Akt in IL-15 increased adaptive immunity is still poorly understood. In this study, we demonstrated that ex vivo IL-15 administration increased BMPC capability of antigen uptake and the expression of costimulatory molecules (such as CD80 and 4-1BB(CD137) ligand [4-1BBL]) and MHC class I molecule via PI3K-Akt pathway. Importantly, PI3K-Akt activity was not only necessary for IL-15 augmented BMPC cross-presentation and CTL priming, but also facilitated IL-15 increased therapeutic potential of the cytolytic capacity and maintenance of BMPC-activated T cells. Thus, these data suggested that PI3K-Akt activity contribute to the superior immune function of IL-15-administrated BMPC and thereby might be therapeutic potential for adaptive immunity.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Células da Medula Óssea/imunologia , Interleucina-15/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Antígenos/metabolismo , Apresentação Cruzada/efeitos dos fármacos , Citosol/metabolismo , Feminino , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologiaRESUMO
Cross-presentation in dendritic cells (DC) requires the endosomal relocations of internalized antigens and the endoplasmic reticulum protein Sec61. Despite the fact that endotoxin-containing pathogen and endotoxin-free antigen have different effects on protein kinase B (Akt) and I-kappa B Kinase α/ß (IKKα/ß) activation, the exact roles of Akt phosphorylation, IKKα or IKKß activation in endotoxin-containing pathogen-derived cross-presentation are poorly understood. In this study, endotoxin-free ovalbumin supplemented with endotoxin was used as a model pathogen. We investigated the effects of endotoxin-containing pathogen and endotoxin-free antigen on Akt phosphorylation, IKKα/ß activation, and explored the mechanisms that the endotoxin-containing pathogen orchestrating the endosomal recruitment of Sec61 of the cross-presentation in bone marrow precursor cells (BMPC). We demonstrated that endotoxin-containing pathogen and endotoxin-free antigen efficiently induced the phosphorylation of Akt-IKKα/ß and Akt-IKKα, respectively. Endotoxin-containing pathogen derived Akt+ IKKα/ß+ Rab5+ signalosome, together with augmented the recruitment of Sec61 toward endosome, lead to the increased cross-presentation in BMPC. Importantly, the endosomal recruitment of Sec61 was partly mediated by the formation of Akt+ IKKα/ß+ signalosome. Thus, these data suggest that Akt+ IKKα/ß+ Rab5+ signalosome contribute to endotoxin-containing pathogen-induced the endosomal recruitment of Sec61 and the superior efficacy of cross-presentation in BMPC.
RESUMO
PURPOSE: To explore the preventive and therapeutic antitumor effects of nicotine-treated immature dendritic cells (imDC). EXPERIMENTAL DESIGN: First, bone marrow-derived imDCs were stimulated with nicotine in vitro, and nicotinic acetylcholine receptor, costimulator molecules, chemokine receptor, and endocytosis ability of imDCs were detected by flow cytometry. Second, the DC-dependent antigen-specific T-cell proliferation, CTL priming, and interleukin-12 secretion were determined by flow cytometry, enzyme-linked immunospot assay, and ELISA, respectively. Finally, preventive and therapeutic antitumor effects of such imDCs were determined by i.p. transfer against tumor challenge or implantation in mice. RESULTS: Nicotine could up-regulate expression of nicotinic acetylcholine receptor, costimulatory molecules, such as CD80, CD86, and CD40, adhesion molecule CD11b, and chemokine receptor CCR7 and enhance endocytosis ability of imDCs. In addition, nicotine could promote imDC-dependent CTL priming and interleukin-12 secretion in vitro. Most importantly, systemic transfer of ex vivo nicotine-stimulated imDCs could reveal preventive and therapeutic effect on tumor development. CONCLUSIONS: Ex vivo nicotine stimulation can significantly improve the efficacy of imDCs for adaptive therapy of cancer and nicotine-treated imDCs may be considered as a potential candidate for preventive and therapeutic tumor vaccination.
Assuntos
Vacinas Anticâncer , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/transplante , Neoplasias Experimentais/terapia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Animais , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Células Dendríticas/imunologia , Endocitose/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunoterapia/métodos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR7 , Receptores de Quimiocinas/efeitos dos fármacos , Receptores de Quimiocinas/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
PYR-41 and thalidomide have therapeutic effects on inflammation-associated diseases with side effects such as tumorigenesis. Cross-presentation allows dendritic cells (DC) to present endogenous antigen and induce protective immunity against microbe infection and tumors. But, up to now, the effects of PYR-41 and thalidomide on cross-presentation are still uncertain. In this study, we investigated the effect and mechanism of PYR-41 and thalidomide on DC cross-presentation by observing Myddosome formation, endosomal recruitment of p97 and Sec61, NF-κB activation, and cross-priming ability. We demonstrated that the inhibition of endosomal recruitment of p97 and Sec61, together with attenuated NF-κB activation and Myddosome formation, contributes to PYR-41- and thalidomide-impaired cross-presentation and thereby reverses cross-activation of T cells. These observations suggest that NF-κB signaling and p97 and Sec61 molecules are candidates for dealing with the side effects of PYR-41 and thalidomide.
Assuntos
Benzoatos/toxicidade , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Furanos/toxicidade , Imunossupressores/toxicidade , Pirazóis/toxicidade , Talidomida/toxicidade , Animais , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Endossomos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Canais de Translocação SEC/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Tumor necrosis factor αinduced protein 8 (TIPE) is highly expressed in many types of malignancies. Apoptosis is the process of programmed cell death which maintains the balance of cell survival and death. TIPE is involved in the carcinogenesis of many tumor types, yet the exact role of TIPE in defective apoptosisassociated carcinogenesis remains uncertain. In the present study, TIPEoverexpressing Raw264.7 and EL4 cells and vector control cells were treated with 4 mJ/cm2 ultraviolet radiation or 2 µg/ml cisplatin. Following ultraviolet irradiation, TIPE overexpression decreased the percentage of apoptotic cells as detected by flow cytometric and reversed the cisplatinmediated decrease in mitochondrial membrane potential by JC1 assay. Western blot analyses also revealed that TIPE overexpression inhibited cisplatininduced activation of caspase3 and 9 and PARP. Secondly, TIPE overexpression increased the levels of phosphorylated JNK, MEK and p38. Moreover, inhibition of JNK and p38, but not MEK, efficiently abolished the cell prosurvival effect of TIPE. Most importantly, an in vivo tumor implantation model revealed that TIPE overexpression augmented the volume and weight of the implanted tumors, indicating that TIPE facilitated tumor formation. We found that TIPE exhibited an antiapoptotic effect via JNK and p38 activation, which ultimately promoted tumor. Hence, the present study revealed that activation of JNK and p38 kinases contribute to the TIPEmediated antiapoptotic effect, indicating that JNK and p38 may be potential therapeutic molecules for TIPE overexpressionassociated diseases.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Cisplatino/farmacologia , MAP Quinase Quinase 4/metabolismo , Neoplasias Experimentais/metabolismo , Transfecção , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Células RAW 264.7RESUMO
Cholinergic anti-inflammatory pathway has therapeutic effect on inflammation-associated diseases. However, the exact mechanism of nicotine-mediated anti-inflammatory effect is still unclear. TIPE2, a new member of tumor necrosis factor-α-induced protein-8 family, is a negative regulator of immune homeostasis. However, the roles of TIPE2 in cholinergic anti-inflammatory effect are still uncertain. Here, we demonstrated that nicotine exerts its anti-inflammatory effect by TIPE2 upregulation and phosphorylated stat3 mediated the inhibition of NF-κB activation, which was supported by the following evidence: firstly, both nicotine and TIPE2 inhibit pro-inflammatory cytokine release via NF-κB inactivation. Secondly, nicotine upregulates TIPE2 expression via α7 nicotinic acetylcholine receptor. Moreover, the enhancement of stat3 phosphorylation and decrease of LPS-induced p65 translocation were achieved by nicotine treatment. Importantly, nicotine treatment augments the interaction of phosphorylated stat3 and p65, indicating that the inhibitory effect of nicotine on NF-κB activation was mediated with protein-protein interactions. Hence, this study revealed that TIPE2 upregulation and stat3 phosphorylation contribute to nicotine-mediated anti-inflammation effect, indicating that TIPE2 and stat3 might be potential molecules for dealing with inflammation-associated diseases.
Assuntos
Inflamação/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Nicotina/administração & dosagem , Fator de Transcrição STAT3/genética , Fator de Transcrição RelA/genética , Animais , Colinérgicos/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , NF-kappa B/genética , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Although the importance of CD4+ T-cell help for generation of an effective CD8+ effector cytotoxic T cell (CTL) response is well established, the role of T-cell help in the activation of memory T cells to become fully functional tumor killer cells is undefined. Using synthetic peptide immunizations corresponding to the major CTLs and T-helper epitopes of ovalbumin, adoptive transfers of ovalbumin-specific memory CTLs (mCTLs), and ovalbumin as the tumor-specific antigen in a mouse tumor model, we have determined that T help is essential for the activation of mCTLs to kill tumors. Our data show that T-helper cells specific for the tumor-associated antigen are required for the reactivation of mCTLs by antigen presented indirectly from tumor. In contrast, effector CTLs do not need T help to kill tumors. These results have implications for induction of tumor immunotherapy by immunization.
Assuntos
Epitopos de Linfócito T/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Proteínas do Ovo/imunologia , Feminino , Memória Imunológica/imunologia , Imunoterapia Adotiva , Linfoma/imunologia , Linfoma/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Ovalbumina/imunologia , Fragmentos de Peptídeos , Células Tumorais CultivadasRESUMO
Hypoxia always exists in the processes involved in the development of lung cancer and contributes to an acidic microenvironment. Despite that hypoxia in the tumor microenvironment is associated with the formation of chemotherapeutic resistance, the exact role of an acidic microenvironment in the development of hypoxia-induced lung cancer multidrug resistance is still unknown. In the present study, we acidized the medium with 2-(N-morpholino)-ethanesulfonic acid (MES monohydrate) to mimic the acidic tumor microenvironment and observed the effects of acidification on lung cancer cell viability, the expression of ATP-binding cassette sub-family G member 2 (ABCG2) and myeloid cell leukemia1 (Mcl-1), and activation of the PI3K-Akt pathway. Thereafter, we investigated the mechanisms involved in the acidification-induced expression of ABCG2 and Mcl-1, and the potential therapeutic strategy to overcome acidification-associated multidrug resistance formation. We demonstrated that acidification obviously increased the expression of ABCG2 and Mcl-1 via PI3KAktmTOR-S6 pathway activation and contributed to multidrug resistance. Inhibition of PI3K-Akt activity efficiently abolished the effect of acidification on cell viability, indicating that the PI3K-Akt pathway may include potential therapeutic target molecules in acidized microenvironment-associated lung cancer chemotherapeutic resistance.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Regulação para Cima/genética , Células A549 , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Resistência a Múltiplos Medicamentos/genética , Humanos , Hipóxia/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Quinases S6 Ribossômicas/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Ativação Transcricional/genética , Microambiente Tumoral/genéticaRESUMO
Despite that ataxia-telangiectasia mutated (ATM) is involved in IL-6 promoted lung cancer chemotherapeutic resistance and metastasis, the exact role of ATM in tumor necrosis factor-alpha (TNF-α) increasing tumor migration is still elusive. In the present study, we demonstrated that TNF-α promoted lung cancer cell migration by up-regulation of matrix metalloproteinase-13 (MMP-13). Notably, by gene silencing or kinase inhibition, we proposed for the first time that ATM is a key up-stream regulator of TNF-α activated ERK/p38-NF-κB pathway. The existence of TNF-α secreted in autocrine or paracrine manner by components of tumor microenvironment highlights the significance of TNF-α in inflammation-associated tumor metastasis. Importantly, in vivo lung cancer metastasis test showed that ATM depletion actually reduce the number of metastatic nodules and cancer nests in lung tissues, verifying the critical role of ATM in metastasis. In conclusion, our findings demonstrate that ATM, which could be activated by lung cancer-associated TNF-α, up-regulate MMP-13 expression and thereby augment tumor metastasis. Therefore, ATM might be a promising target for prevention of inflammation-associated lung cancer metastasis.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células A549 , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Inativação Gênica , Humanos , Inflamação , Leucócitos Mononucleares/citologia , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , NF-kappa B/metabolismo , Metástase Neoplásica , RNA Interferente Pequeno/metabolismo , Baço/citologia , Regulação para CimaRESUMO
Cross-presentation by dendritic cells (DCs) requires surface molecules such as lectin, CD40, langerin, heat shock protein, mannose receptor, mediated endocytosis, the endosomal translocation of internalized antigen, and the relocation of transporter associated with antigen processing (TAP). Although the activation of α7 nicotinic acetylcholine receptor (α7 nAchR) up-regulate surface molecule expression, augment endocytosis, and enhance cross-presentation, the molecular mechanism of α7 nAchR activation-increased cross-presentation is still poorly understood. In this study, we investigated the role of mannose receptor in nicotine-increased cross-presentation and the mechanism that endotoxins orchestrating the recruitment of TAP toward endosomes. We demonstrated that nicotine increase the expressiones of mannose receptor and Toll-like receptor 4 (TLR4) via PI3K-Akt-mTOR-p70S6 pathway. Both endosomal translocation of mannose receptor-internalized antigens and TLR4 sig- naling are necessary for nicotine-augmented cross-presentation and cross-priming. Importantly, the recruitment of TAP toward endosomes via TLR4-MyD88-IRAK4 signaling contributes to nicotine-increased cross-presentation and cross-activation of T cells. Thus, these data suggest that increased recruitment of TAP to Ag-containing vesicles contributes to the superior cross-presentation efficacy of α7 nAchR activated DCs.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Dendríticas/metabolismo , Endossomos/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Nicotina/farmacologia , Receptores de Superfície Celular/metabolismo , Receptor 4 Toll-Like/metabolismo , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação Cruzada , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Humanos , Ativação Linfocitária , Receptor de Manose , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Our previous studies show that the phosphorylation of ataxia-telangiectasia mutated (ATM) induced by interleukin 6 (IL-6) treatment contributes to multidrug resistance formation in lung cancer cells, but the exact role of ATM activation in IL-6 increased metastasis is still elusive. In the present study, matrix metalloproteinase-3 (MMP-3) and MMP-13 were firstly demonstrated to be involved in IL-6 correlated cell migration. Secondly, IL-6 treatment not only increased MMP-3/MMP-13 expression but also augmented its activities. Thirdly, the inhibition of ATM phosphorylation efficiently abolished IL-6 up-regulating MMP-3/MMP-13 expression and increasing abilities of cell migration. Most importantly, the in vivo test showed that the inhibition of ATM abrogate the effect of IL-6 on lung cancer metastasis via MMP-3/MMP-13 down-regulation. Taken together, these findings demonstrate that IL-6 inducing ATM phosphorylation increases the expression of MMP-3/MMP-13, augments the abilities of cell migration, and promotes lung cancer metastasis, indicating that ATM is a potential target molecule to overcome IL-6 correlated lung cancer metastasis.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Neoplasias Pulmonares/secundário , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor necrosis factor-α-induced protein 8-like 2 (TNFAIP8L2, TIPE2), which belongs to the TNF-α-induced protein 8 family, is a negative regulator of immune homeostasis. Although pro-inflammatory cytokines such as TNF-α have been reported to be involved in liver carcinoma metastasis, the effect of TIPE2 on hepatocellular carcinoma metastasis remains unknown. We demonstrate that TNF-α clearly augments MMP-13/MMP-3 expression and promotes cell migration in HepG2 cells through activation of the Erk1/2-NF-κB pathways. Interestingly, in addition to human PBLs, macrophages and fibroblasts, liver cancer cells specifically express TNF-α following LPS treatment. Most importantly, TIPE2 overexpression efficiently abrogates the effects of LPS on TNF-α secretion and abolishes the effects of TNF-α on MMP-13/MMP-3 upregulation, cell migration and Erk1/2-NF-κB activation. Taken together, these findings demonstrate that TIPE2 was able to suppress TNF-α-induced hepatocellular carcinoma metastasis by inhibiting Erk1/2 and NF-κB activation, indicating that both TNF-α and TIPE2 might be potential targets for the treatment of HCC metastasis.
Assuntos
Carcinoma Hepatocelular/patologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Hepáticas/patologia , Fator de Necrose Tumoral alfa/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , TransfecçãoRESUMO
Our previous studies showed that α7 nicotinic acetylcholine receptor (nAchR) agonist nicotine has stimulatory effects on murine bone marrow-derived semimature DCs, but the effect of nicotine on peripheral blood mononuclear cell- (PBMC-) derived human semimature dendritic cells (hu-imDCs) is still to be clarified. In the present study, hu-imDCs (cultured 4 days) were conferred with ex vivo lower dose nicotine stimulation and the effect of nicotine on surface molecules expression, the ability of cross-presentation, DCs-mediated PBMC priming, and activated signaling pathways were determined. We could demonstrate that the treatment with nicotine resulted in increased surface molecules expression, enhanced hu-imDCs-mediated PBMC proliferation, upregulated release of IL-12 in the supernatant of cocultured DCs-PBMC, and augmented phosphorylation of Akt and ribosomal protein S6. Nicotine associated with traces of LPS efficiently enhanced endosomal translocation of internalized ovalbumin (OVA) and increased TAP-OVA colocalization. Importantly, the upregulation of nicotine-increased surface molecules upregulation was significantly abrogated by the inhibition of Akt kinase. These findings demonstrate that ex vivo nicotine stimulation augments hu-imDCs surface molecules expression via Akt-S6 pathway, combined with increased Ag-presentation result in augmented efficacy of DCs-mediated PBMC proliferation and Th1 polarization.