RESUMO
Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.
Assuntos
Neoplasias da Mama , Nucleotídeos de Guanina , Humanos , Feminino , Nucleotídeos de Guanina/metabolismo , Nucleotídeos de Guanina/farmacologia , Guanosina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Guanosina Trifosfato/farmacologia , Adenosina/farmacologia , Adenosina/metabolismo , DipiridamolRESUMO
There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.
Assuntos
Anti-Inflamatórios , Fluoxetina , Interleucina-6 , Relação Estrutura-Atividade , Animais , Fluoxetina/farmacologia , Camundongos , Interleucina-6/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Citocinas/metabolismo , Receptor 3 Toll-Like/metabolismo , Poli I-C/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/química , Inflamação/tratamento farmacológicoRESUMO
Residual cancer cells after radiation therapy may acquire malignant phenotypes such as enhanced motility and migration ability, and therefore it is important to identify targets for preventing radiation-induced malignancy in order to increase the effectiveness of radiotherapy. G-Protein-coupled receptors (GPCRs) such as adenosine A2B receptor and cannabinoid receptors (CB1, CB2, and GPR55) may be involved, as they are known to have roles in proliferation, invasion, migration and tumor growth. In this study, we investigated the involvement of A2B and cannabinoid receptors in γ-radiation-induced enhancement of cell migration and actin remodeling, as well as the involvement of cannabinoid receptors in cell migration enhancement via activation of A2B receptor in human lung cancer A549 cells. Antagonists or knockdown of A2B, CB1, CB2, or GPR55 receptor suppressed γ-radiation-induced cell migration and actin remodeling. Furthermore, BAY60-6583 (an A2B receptor-specific agonist) enhanced cell migration and actin remodeling in A549 cells, and this enhancement was suppressed by antagonists or knockdown of CB2 or GPR55, though not CB1 receptor. Our results indicate that A2B receptors and cannabinoid CB1, CB2, and GPR55 receptors all contribute to γ-radiation-induced acquisition of malignant phenotypes, and in particular that interactions of A2B receptor and cannabinoid CB2 and GPR55 receptors play a role in promoting cell migration and actin remodeling. A2B receptor-cannabinoid receptor pathways may be promising targets for blocking the appearance of malignant phenotypes during radiotherapy of lung cancer.
Assuntos
Canabinoides , Neoplasias Pulmonares , Humanos , Células A549 , Actinas , Canabinoides/farmacologia , Canabinoides/metabolismo , Neoplasias Pulmonares/radioterapia , Receptor A2B de Adenosina , Receptores de CanabinoidesRESUMO
Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.
Assuntos
Anti-Inflamatórios/farmacologia , Cromonas/farmacologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor 4 Toll-Like/metabolismo , Trimebutina/farmacologia , Animais , Anti-Inflamatórios/química , Cromonas/química , Proteína HMGB1/metabolismo , Humanos , Camundongos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Trimebutina/químicaRESUMO
Glioblastoma is the most common malignant tumor of the central nervous system and is treated with a combination of surgery, radiation and chemotherapy. However, the tumor often acquires radiation resistance, which is characterized by an increased DNA damage response (DDR). Here, we show that CD73, which generates extracellular adenosine from ATP, and A2B receptor, which is activated by adenosine, are involved in the γ-radiation-induced DDR and the enhanced migration ability of human glioblastoma cell line A172. To investigate DDR, we evaluated ataxia telangiectasia mutated (ATM) activation and focus formation of histone H2A isoform γ (γH2AX) and p53-binding protein 1 (53BP1) in the nucleus of A172 cells after γ-irradiation. Antagonists of A2B receptor and CD73, or knockdown with small interfering RNA (siRNA), suppressed γ-radiation-induced DDR and promoted γ-radiation-induced cell death, as well as suppressing γ-radiation-induced cell migration and actin remodeling. These results suggest that activation of A2B receptor by extracellular adenosine generated via CD73 promotes γ-radiation-induced DDR, leading to recovery from DNA damage, and also enhances cell migration and actin remodeling. The CD73-A2B receptor pathway may be a promising target for overcoming radiation resistance and the acquisition of malignant phenotypes during radiotherapy of glioblastoma.
Assuntos
5'-Nucleotidase/metabolismo , Reparo do DNA/efeitos da radiação , Glioblastoma/radioterapia , Tolerância a Radiação/genética , Receptor A2B de Adenosina/metabolismo , 5'-Nucleotidase/genética , Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/efeitos da radiação , Quimiorradioterapia/métodos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Tolerância a Radiação/efeitos dos fármacosRESUMO
Radiation is an effective cancer treatment, but cancer cells can acquire radioresistance, which is associated with increased DNA damage response and enhanced proliferative capacity, and therefore, it is important to understand the intracellular biochemical responses to γ-irradiation. The transient receptor potential melastatin 8 (TRPM8) channel plays roles in the development and progression of tumors, but it is unclear whether it is involved in the DNA damage response induced by γ-irradiation. Here, we show that a TRPM8 channel inhibitor suppresses the DNA damage response (phosphorylated histone variant H2AX-p53-binding protein 1 (γH2AX-53BP1) focus formation) and colony formation of B16 melanoma cells. Furthermore, the TRPM8 channel-specific agonist WS-12 enhanced the DNA damage response and increased the survival fraction after γ-irradiation. We found that the TRPM8 channel inhibitor enhanced G2/M phase arrest after γ-irradiation. Phosphorylation of ataxia telangiectasia mutated and p53, which both contribute to the DNA damage response was also suppressed after γ-irradiation. In addition, the TRPM8 channel inhibitor enhanced the γ-irradiation-induced suppression of tumor growth in vivo. We conclude that the TRPM8 channel is involved in radiation-induced DNA damage repair and contributes to the radioresistance of B16 melanoma cells. TRPM8 channel inhibitors might be clinically useful as radiosensitizers to enhance radiation therapy of melanoma.
Assuntos
Dano ao DNA , Reparo do DNA , Melanoma Experimental/radioterapia , Canais de Cátion TRPM/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Anilidas/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Raios gama , Histonas/metabolismo , Masculino , Melanoma Experimental/metabolismo , Proteínas de Membrana/metabolismo , Mentol/análogos & derivados , Mentol/farmacologia , Camundongos , Fosforilação , Radiossensibilizantes/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced in response to endoplasmic reticulum (ER) stress induced by various stressors, but its involvement in the radioresistance of tumor cells is unknown. Here, we found that MANF is released after γ-irradiation (2 Gy and 4 Gy) of B16 melanoma cells, and its release was suppressed by 4-phenylbutyric acid, an ER stress inhibitor. MANF was not released after low-dose (1 Gy) γ-irradiation, but pretreatment of 1 Gy-irradiated cells with recombinant MANF enhanced the cellular DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the DNA damage response and p53 activation by γ-irradiation (2 Gy) were suppressed, and reproductive cell death was increased. MANF also activated the ERK signaling pathway. Our findings raise the possibility that MANF could be a new target for overcoming radioresistance.
Assuntos
Estresse do Retículo Endoplasmático/efeitos da radiação , Retículo Endoplasmático/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Fatores de Crescimento Neural/genética , Tolerância a Radiação/genética , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Raios gama , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/radioterapia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/metabolismo , Fenilbutiratos/farmacologia , Fosforilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Agents that promote DNA repair may be useful as radioprotectants to minimize side effects such as radiation pneumonia caused by damage to normal cells during radiation therapy to treat lung cancer. We have reported that extracellular nucleotides and nucleosides are involved in the P2 or P1 receptor-mediated DNA damage response (DDR) after γ-irradiation. Here, we investigated the effects of ATP, UTP, GTP, ITP and their metabolites on the γH2AX/53BP1 focus formation in nuclei (a measure of γ-irradiation-induced DDR) and the survival of γ-irradiated immortalized human bronchial epithelial (BEAS-2B) cells. Fluorescence immunostaining showed that ATP and ADP increase DDR and DNA repair, and exhibit radioprotective effects as evaluated by colony formation assay. These effects of ATP or ADP were blocked by inhibitors of P2X7 or P2Y12 receptor, respectively, and by ERK1/2 inhibitor. ATP and ADP enhanced phosphorylation of ERK1/2 by suppressing MKP-1 and MKP-3 expression after γ-irradiation. These results indicate that ATP and ADP exhibit radioprotective effects by phosphorylation of ERK1/2 via activation of P2X7 and P2Y12 receptors, respectively, to promote γ-irradiation-induced DDR and DNA repair. ATP and ADP appear to be candidates for radioprotectants to reduce damage to non-cancerous cells during lung cancer radiotherapy by promoting DDR and DNA repair.
Assuntos
Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/efeitos da radiação , Raios gama , Agonistas do Receptor Purinérgico P2X/farmacologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Protetores contra Radiação/farmacologia , Receptores Purinérgicos P2X7/efeitos dos fármacos , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaio de Unidades Formadoras de Colônias , Dano ao DNA/efeitos da radiação , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , FosforilaçãoRESUMO
It is therapeutically important to elucidate the factors involved in the radiation resistance of tumors. We previously showed that ATP is released from mouse melanoma B16 cells in response to γ-irradiation, but the role of adenosine, a metabolite of ATP, is still unclear. Here, we show that the adenosine A2B receptor is involved in DNA damage repair and radioresistance in mouse melanoma B16 cells. The DNA damage response after γ-irradiation was attenuated by pretreatment with A2B receptor antagonists, such as PSB603, while it was enhanced by pretreatment with A2B receptor agonists, such as BAY60-6583. γ-Irradiation decreased the cell survival rate, and pretreatment with PSB603 further reduced the survival rate. On the other hand, pretreatment with BAY60-6583 increased the cell survival rate after irradiation. The DNA damage response and the cell survival rate after γ-irradiation were both decreased in A2B-knockdown cells. In vivo experiments in mice confirmed that tumor growth was suppressed and delayed in the irradiated group pretreated with PSB603, compared with the irradiation-alone group. Our results indicate that adenosine A2B receptor contributes to radioresistance, and could be a new target for the development of agents to increase the efficacy of radiotherapy.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Dano ao DNA/efeitos dos fármacos , Tolerância a Radiação/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Raios gama/uso terapêutico , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Radiossensibilizantes , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Magnolia Flower is a crude drug used for the treatment of headaches, toothaches, and nasal congestion. Here, we focused on Magnolia kobus, one of the botanical origins of Magnolia Flower, and collected the flower parts at different growth stages to compare chemical compositions and investigate potential inhibitory activities against interleukin-2 (IL-2) production in murine splenic T cells. After determining the structures, we examined the inhibitory effects of the constituents of the bud, the medicinal part of the crude drug, against IL-2 production. We first extracted the flower parts of M. kobus from the bud to fallen bloom stages and analysed the chemical compositions to identify the constituents characteristic to the buds. We found that the inhibitory activity of the buds against IL-2 production was more potent than that of the blooms. We isolated two known compounds, tiliroside (1) and syringin (2), characteristic to the buds from the methanol (MeOH) extract of Magnolia Flower. Moreover, we examined the inhibitory activities of both compounds against IL-2 production and found that tiliroside (1) but not syringin (2), showed strong inhibitory activity against IL-2 production and inhibited its mRNA expression. Thus, our strategy to examine the relationship between chemical compositions and biological activities during plant maturation could not only contribute to the scientific evaluation of medicinal parts of crude drugs but also assist in identifying biologically active constituents that have not yet been reported.
Assuntos
Interleucina-2/metabolismo , Magnolia/química , Extratos Vegetais/química , Animais , Linhagem Celular , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Flores/química , Flores/metabolismo , Glucosídeos/química , Glucosídeos/isolamento & purificação , Glucosídeos/farmacologia , Interleucina-2/genética , Magnolia/metabolismo , Camundongos , Fenilpropionatos/química , Fenilpropionatos/isolamento & purificação , Fenilpropionatos/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.
Assuntos
Adenocarcinoma/tratamento farmacológico , Claudina-1/genética , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Doxorrubicina/efeitos adversos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Esferoides Celulares/efeitos dos fármacos , Quinase Induzida por NF-kappaBRESUMO
Transient receptor potential (TRP) channels are a family of non-selective cation channels that are functionally expressed in various organs and cells. Among them, transient receptor potential vanilloid (TRPV) 1 and TRPV4 channels are expressed in T cells, where they serve as Ca2+ channels for T-cell receptor signaling [Bertin et al., 2014, Majhi et al., 2015]. Here, we show that not only TRPV1 and TRPV4 channel inhibitors, but also a transient receptor potential melastatin (TRPM) 8 channel inhibitor can suppress murine T-cell activation. Mouse splenic lymphocytes pretreated with N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 channel-selective inhibitor, showed significantly reduced IL-2 and IL-6 release from T cells after stimulation with anti-CD3ε/anti-CD28 antibodies or concanavalin A. AMTB also suppressed IL-2 mRNA expression and activation of extracellular signal-regulated kinase 1/2, which is involved in IL-2 production. Further, the increase of CD25 (IL-2 receptor alpha chain) expression after T-cell activation was suppressed by AMTB. TRPM8 channel was expressed in CD4+ T cells isolated from splenocytes, and we confirmed that the release of IL-2 from isolated CD4+ T cells was significantly suppressed by AMTB. In vitro re-stimulation of splenocytes from external antigen-immunized mice with the same antigen induced IL-2 and IL-6 production, which was significantly suppressed by AMTB. Thus, the TRPM8 channel inhibitor AMTB suppresses T-cell activation induced by various stimulants.
Assuntos
Antígenos/metabolismo , Benzamidas/farmacologia , Concanavalina A/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Interleucina-2/biossíntese , Interleucina-6/biossíntese , Camundongos , Baço/citologiaRESUMO
Damaged tissues and cells release intracellular purine nucleotides, which serve as intercellular signaling factors. We previously showed that exogenously added adenine nucleotide (250⯵M ATP) suppressed the activation of murine splenic T lymphocytes. Here, we examined the effects of other purine nucleotides/nucleosides on mouse T cell activation. First, we found that pretreatment of mouse spleen T cells with 250⯵M GTP, GDP, GMP, guanosine, ITP, IDP, IMP or inosine significantly reduced the release of stimulus-inducible cytokine IL-2. This suppression of IL-2 release was not caused by induction of cell death. Further studies with GTP, ITP, guanosine and inosine showed that pretreatment with these nucleotides/nucleosides also suppressed release of IL-6. However, these nucleotides/nucleosides did not suppress stimulus-induced phosphorylation of ERK1/2, suggesting that the suppression of the release of inflammatory cytokines does not involve inhibition of ERK1/2 signaling. In contrast to ATP pretreatment at the same concentration, guanine or inosine nucleotides/nucleosides did not attenuate the expression of CD25. Our findings indicate that exogenous guanine or inosine nucleotides/nucleosides can suppress inflammatory cytokine release from T cells, and may be promising candidates for use as supplementary agents in the treatment of T cell-mediated immune diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Nucleotídeos de Guanina/farmacologia , Guanosina/farmacologia , Nucleotídeos de Inosina/farmacologia , Inosina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Interleucina-2/antagonistas & inibidores , Interleucina-2/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologiaRESUMO
External stimuli, such as radiation, induce inflammatory cytokine and chemokine production in skin, but the mechanisms involved are not completely understood. We previously showed that the P2Y11 nucleotide receptor, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) all participate in interleukin (IL)-6 production induced by γ-irradiation. Here, we focused on the transient receptor potential vanilloid 4 (TRPV4) channel, which is expressed in skin keratinocytes and has been reported to play a role in inflammation. We found that irradiation of human epidermal keratinocytes HaCaT cells with 5 Gy of γ-rays (137Cs: 0.75 Gy/min) induced IL-6 and IL-8 production. HaCaT cells treated with TRPV4 channel agonist GSK1016790A also showed increased IL-6 and IL-8 production. In both cases, IL-6/IL-8 production was not increased at 24 h after stimulation, but was increased at 48 h. ATP was released from cells exposed to γ-irradiation or TRPV4 channel agonist, and the release was suppressed by TRPV4 channel inhibitors. The γ-irradiation-induced increase in IL-6 and IL-8 production was suppressed by apyrase (ecto-nucleotidase), NF157 (selective P2Y11 receptor antagonist) and SB203580 (p38 MAPK inhibitor). GSK1016790A-induced inhibitor of kappa B-alpha (IκBα) decomposition, which causes NF-κB activation was suppressed by NF157 and SB203580, and γ-irradiation-induced IκBα decomposition was suppressed by TRPV4 channel inhibitors. Our results suggest that γ-irradiation of keratinocytes induces ATP release via activation of the TRPV4 channel, and then ATP activates P2Y11 receptor and p38 MAPK-NF-κB signaling, resulting in IL-6/IL-8 production.
Assuntos
Trifosfato de Adenosina/metabolismo , Raios gama , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Queratinócitos/metabolismo , Canais de Cátion TRPV/fisiologia , Trifosfato de Adenosina/efeitos da radiação , Linhagem Celular Transformada , Epiderme/metabolismo , Epiderme/efeitos da radiação , Humanos , Interleucina-6/efeitos da radiação , Interleucina-8/efeitos da radiação , Queratinócitos/efeitos da radiação , Canais de Cátion TRPV/efeitos da radiaçãoRESUMO
Skin inflammation is caused by excessive production of cytokines and chemokines in response to an external stimulus, such as radiation, but the mechanisms involved are not completely understood. Here, we report a novel mechanism of γ-irradiation-induced interleukin-6 (IL-6) production mediated by P2Y11 receptors in epidermal cells. After irradiation of HaCaT cells derived from human epidermal keratinocytes with 5 Gy of γ-rays (137Cs: 0.78 Gy/min), IL-6 production was unchanged at 24 h after γ-irradiation, but was increased at 48 h. IL-6 mRNA was increased at 30 h, and IL-6 production was increased at 33 h after irradiation. The production of IL-6 was sustained at least for 4 d after irradiation. P2Y11 receptor antagonist NF157 inhibited IL-6 production in irradiated cells. Treatment with ATP, a ligand of P2Y11 receptor caused IL-6 production within 24 h. ATP-induced IL-6 production was also suppressed by NF157. Extracellular ATP level was increased after irradiation. The p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling was involved in the production of IL-6 at the downstream of P2Y11 receptor activation. In addition, the cell cycle was arrested at the G2/M phase, and DNA repair foci were not disappeared at 48 h after γ-irradiation. The protein level of histone methylation enzyme G9a, which inhibits IL-6 production, was decreased after γ-irradiation. In conclusion, we suggest that γ-irradiation induces sustained IL-6 production in HaCaT cells from 33 h after irradiation, which is mediated through P2Y11 receptor-p38 MAPK-NF-κB signaling pathway and G9a degradation. This is a novel mechanism of cytokine production in γ-irradiated cells.
Assuntos
Raios gama , Interleucina-6/metabolismo , Queratinócitos/efeitos da radiação , Receptores Purinérgicos P2/metabolismo , Linhagem Celular , Dano ao DNA , Células Epidérmicas , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos da radiação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Sepsis is a systemic inflammatory response syndrome triggered by lipopolysaccharide (LPS), an outer membrane component of gram-negative bacteria, and cytokine production via LPS-induced macrophage activation is deeply involved in its pathogenesis. Effective therapy of sepsis has not yet been established. However, it was reported that transient receptor potential vanilloid 1 (TRPV1) channel antagonist capsazepine (CPZ; a capsaicin analogue) attenuates sepsis in a murine model [Ang et al., PLoS ONE 6(9) (2011) e24535; J. Immunol. 187 (2011) 4778-4787]. Here, we profiled the effects of four TRPV1 channel antagonists, AMG9810, SB366791, BCTC and CPZ, on the release of IL-6, IL-1ß and IL-18, and on expression of cyclooxygenase 2 (COX-2) in LPS-activated macrophages. Treatment of murine macrophage J774.1 cells or BALB/c mouse-derived intraperitoneal immune cells with LPS induced pro-inflammatory cytokines production and COX-2 expression. Pretreatment with AMG9810 or CPZ significantly suppressed the release of IL-6, IL-1ß and IL-18, and COX-2 expression, whereas SB366791 and BCTC were less effective. These results support a role of TRPV1 channel in macrophage activation, but also indicate that only a subset of TRPV1 channel antagonists may be effective in suppressing inflammatory responses. These results suggest that at least some TRPV1 channel antagonists, such as AMG9810 and CPZ, may be candidate anti-inflammatory agents for treatment of sepsis.
Assuntos
Anti-Inflamatórios/administração & dosagem , Ciclo-Oxigenase 2/imunologia , Citocinas/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Canais de Cátion TRPV/antagonistas & inibidores , Anti-Inflamatórios/classificação , Linhagem Celular , Células , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Canais de Cátion TRPV/imunologiaRESUMO
Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.
Assuntos
Raios gama/uso terapêutico , Melanoma Experimental/terapia , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Radiossensibilizantes/uso terapêutico , Receptores Purinérgicos P2X7/metabolismo , Corantes de Rosanilina/uso terapêutico , Animais , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Humanos , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Antagonistas do Receptor Purinérgico P2X/farmacologia , Radiossensibilizantes/farmacologia , Receptores Purinérgicos P2X7/genética , Corantes de Rosanilina/farmacologia , Carga Tumoral/efeitos dos fármacosRESUMO
Radiosensitizers are used in cancer therapy to increase the γ-irradiation susceptibility of cancer cells, including radioresistant hypoxic cancer cells within solid tumors, so that radiotherapy can be applied at doses sufficiently low to minimize damage to adjacent normal tissues. Radiation-induced DNA damage is repaired by multiple repair systems, and therefore these systems are potential targets for radiosensitizers. We recently reported that the transient receptor potential vanilloid type 1 (TRPV1) channel is involved in early responses to DNA damage after γ-irradiation of human lung adenocarcinoma A549 cells. Therefore, we hypothesized that TRPV1 channel inhibitors would have a radiosensitizing effect by blocking repair of radiation-induced cell damage. Here, we show that pretreatment of A549 cells with the TRPV1 channel inhibitors capsazepine, AMG9810, SB366791 and BCTC suppressed the γ-ray-induced activation of early DNA damage responses, i.e., activation of the protein kinase ataxia-telangiectasia mutated (ATM) and accumulation of p53-binding protein 1 (53BP1). Further, the decrease of survival fraction at one week after γ-irradiation (2.0 Gy) was enhanced by pretreatment of cells with these inhibitors. On the other hand, inhibitor pretreatment did not affect cell viability, the number of apoptotic or necrotic cells, or DNA synthesis at 24 h after irradiation. These results suggest that inhibition of DNA repair by TRPV1 channel inhibitors in irradiated A549 cells caused gradual loss of proliferative ability, rather than acute facilitation of apoptosis or necrosis. TRPV1 channel inhibitors could be novel candidates for radiosensitizers to improve the efficacy of radiation therapy, either alone or in combination with other types of radiosensitizers.
Assuntos
Raios gama , Radiossensibilizantes/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Acrilamidas/farmacologia , Anilidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cinamatos/farmacologia , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Necrose/induzido quimicamente , Neoplasias/metabolismo , Pirazinas/farmacologia , Piridinas/farmacologia , Canais de Cátion TRPV/metabolismoRESUMO
Kupffer cells, which are resident macrophages in liver, can produce various cytokines and chemokines that induce hepatitis and liver fibrosis. It is suggested that extracellular ATP-induced activation of macrophage P2X7 receptor plays an important role in inflammation via release of pro-inflammatory mediators, but the role of P2X7 receptor in Kupffer cells remains unclear. Here, we show that activation of P2X7 receptor in Kupffer cells causes multiple inflammatory responses, using the clonal mouse Kupffer cell line (KUP5) that we previously established. Treatment of LPS-primed Kupffer cells with 3 mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of MAPK, cell lysis, IL-1ß release, prostaglandin E2 (PGE2) release, high mobility group box1 (HMGB1) release, and major histocompatibility complex (MHC) class I shedding. These events were significantly suppressed by pretreatment with P2X7 antagonist A438079, indicating involvement of P2X7 receptor activation in these inflammatory responses. Our results suggest that extracellular ATP-induced activation of P2X7 receptor of Kupffer cells plays multiple roles in the inflammatory response in liver. P2X7 receptor might be a new therapeutic target for treatment of liver diseases.
Assuntos
Trifosfato de Adenosina/imunologia , Dinoprostona/imunologia , Proteína HMGB1/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interleucina-1beta/imunologia , Células de Kupffer/imunologia , Receptores Purinérgicos P2X7/imunologia , Animais , Células Cultivadas , Inflamação/imunologia , Células de Kupffer/citologia , Camundongos , Camundongos Endogâmicos C57BL , NecroseRESUMO
Recent studies suggest the effect of radiation is observed not only in irradiated cells but also in adjacent non-irradiated cells (bystander effect), although the mechanism has not yet been fully revealed. This bystander effect may be caused by intercellular communication via a gap junction or by messengers released from irradiated cells, such as reactive oxygen species, nitric oxide, or cytokines. However, an unknown mechanism is also possible in the bystander effect. On the other hand, it is known that extracellular ATP, ADP, uridine 5'-triphosphate (UTP), and uridine 5'-diphosphate (UDP), which are released from cells, act as intercellular signaling molecules by activating purinergic P2X and P2Y receptors (purinergic signaling). Recently, I have suggested these extracellular nucleotides may be novel mediators of a radiation-induced bystander effect, because our recent studies indicated that purinergic signaling is involved in important cellular responses to radiation. Our data indicate that ionizing irradiation causes activation of the transient receptor potential melastatin type 2 (TRPM2) channel, and then ATP is released from cells through the anion channel or connexin43 hemichannel mediated by the activation of a P2X7 receptor. The released nucleotides activate P2Y6 and P2Y12 receptors, which are involved in the DNA damage response after irradiation. Activation of the P2Y6 receptor is also involved in radiation-induced activation of the epithelial growth factor receptor-extracellular signal regulated protein kinase (EGFR-ERK)1/2 pathway and subsequent nuclear translocation of EGFR, which plays a role in DNA repair. Further, the induction of an antioxidant after irradiation is also mediated by the activation of the P2Y receptor. In conclusion, purinergic signaling could play an important role in the protective cellular response to ionizing irradiation.