RESUMO
BACKGROUND: Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function. METHODS: We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays. RESULTS: PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function. CONCLUSIONS: The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Fosfolipase C gama/biossíntese , Fosfolipase C gama/genética , Doença de Alzheimer/patologia , Animais , Feminino , Variação Genética/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: Interleukin 2 (IL-2) influences the development and severity of pain due to its antinociceptive and immunomodulatory effects. Its production is influenced by the increased expression of c-Cbl (Casitas B-lineage lymphoma proto-oncogene) and Cbl-b E3 ubiquitin ligases. We evaluated the effects on IL-2-mediated changes in c-Cbl and Cbl-b expression in a rat model of chronic neuropathic pain. METHODS: Peripheral neuropathy was induced in adult male Sprague-Dawley rats weighing 250-300 g by chronic spinal nerve ligation. Half of the spinal cord ipsilateral to the nerve injury was harvested at 1, 3, and 6 weeks, and the expression levels of IL-2, c-Cbl, Cbl-b, phospholipase C-γ1 (PLC-γ1), ZAP70, and protein kinase Cθ (PKCθ), as well as ubiquitin conjugation, were evaluated. RESULTS: Total IL-2 mRNA levels were significantly decreased at 3 and 6 weeks after nerve injury compared to those in sham-operated rats. The mRNA levels of c-Cbl and Cbl-b, as well as the level of ubiquitin conjugation, were significantly increased at 3 and 6 weeks. In contrast, the levels of phosphorylated ZAP70 and PLC-γ1 were decreased at 3 and 6 weeks after spinal nerve ligation. Ubiquitination of PLC-γ1 and PKCθ was increased at 3 and 6 weeks. CONCLUSIONS: Our results suggest that ubiquitin and the E3 ubiquitin ligases c-Cbl and Cbl-b function as neuroimmune modulators in the subacute phase of neuropathic pain after nerve injury.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Interleucina-2/biossíntese , Doenças do Sistema Nervoso Periférico/metabolismo , Proteínas Proto-Oncogênicas c-cbl/biossíntese , Ubiquitina-Proteína Ligases/biossíntese , Animais , Ligadura , Masculino , Fosfolipase C gama/biossíntese , Proteína Quinase C-theta/biossíntese , Ratos , Medula Espinal/metabolismo , Nervos Espinhais/lesões , Ubiquitina/metabolismo , Ubiquitinação , Proteína-Tirosina Quinase ZAP-70/biossínteseRESUMO
The management of hepatic failure is undoubtedly difficult, and poor results have led to the search for novel therapeutic approaches. Nowadays, anti-apoptotic gene therapy is considered as an ideal approach. It has been proved that phospholipase Cγ2 (PLCγ2) is involved in the apoptosis of immune cells and tumor cells; however, whether this gene is related to hepatocyte death is still unclear. This study examined the role of PLCγ2 by inhibiting its expression in rat hepatocytes with siRNA. We also further analyzed the cellular mechanism by which the expression inhibition of PLCγ2 induces cell death. Silencing PLCγ2 gene by adenovirus vector expressing PLCγ2-targeted siRNA caused the great decline in the number of G1- and G2/M phase cells, the significant increase in the number of S phase cells, and the obvious reduction in apoptosis index. In addition, silencing PLCγ2 gene relieved the rat hepatocyte damage, such as the cell shrinkage and chromatin condensation, nuclear fragmentation. Further analysis of Ad-PLCγ2 siRNA-transfected hepatocytes demonstrated that suppression of PLCγ2 gene expression could cause the caspase dependent cell death by inhibiting the signal pathway MEKK1/MKK4/JNK1/2/c-Jun. In conclusion, these findings suggest that interference with PLCγ2 expression could relieve the inhibitory effect of PLCγ2 on hepaocyte apoptosis, thus, promote proliferation through inactivating PKCδ-mediated JNK1/2 signaling pathway.
Assuntos
Apoptose , Ciclo Celular , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Hepatócitos/enzimologia , Sistema de Sinalização das MAP Quinases , Fosfolipase C gama/biossíntese , Animais , Hepatócitos/citologia , Fosfolipase C gama/genética , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Protection of the gastric mucosa from acute lesions induced by various irritants is a pertinent issue in the field of critical care medicine. In this study, we investigated the gastroprotective effects of astragaloside IV on acute gastric lesions in rats under stressful conditions. METHODS: Rats were randomized into six groups. Group 1 and 2 received 10% Tween 80 (vehicle). Group 3 received 20 mg/kg of omeprazole, a proton pump inhibitor. Groups 4, 5 and 6 received astragaloside IV at concentration of 1, 10, and 50 mg/kg, respectively. As a means to induce gastric lesions, Groups 2-6 were subjected to water immersion and restraint stress for 12 hours after treatment. RESULTS: Our present studies show that compared to rats in group 2, treatment with 1 to 50 mg/kg astragaloside IV significantly decreased the size of gastric lesions, MDA, TNFα and MCP1 levels, in addition to normalizing gastric pH, gastric mucus and SOD levels (P<0.05). Histomorphological examination confirmed that treatment with astragaloside IV elicited a dosage-dependent protective effect on the gastric mucosa. Furthermore, pretreatment with astragaloside IV resulted in significant elevations in HSP70 and reduction in Bax, along with over-expression of PLCγ response level, which was further confirmed via immunohistochemical analysis. CONCLUSIONS: The acute gastric lesions induced are attenuated by pretreatment with astragaloside IV which is possibly due to the enhancing of the expression of HSP70 with concomitant antioxidant, anti-inflammatory and anti-apoptotic capacity.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Mucosa Gástrica/metabolismo , Saponinas/uso terapêutico , Úlcera Gástrica/tratamento farmacológico , Triterpenos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Mucosa Gástrica/lesões , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , Malondialdeído/metabolismo , Omeprazol/efeitos adversos , Fosfolipase C gama/biossíntese , Ratos , Ratos Sprague-Dawley , Úlcera Gástrica/induzido quimicamente , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
In the absence of extracellular stimulation the adaptor protein growth factor receptor-bound protein (Grb2) and the phospholipase Plcγ1 compete for the same binding site on fibroblast growth factor receptor 2 (FGFR2). Reducing cellular Grb2 results in upregulation of Plcγ1 and depletion of the phospholipid PI(4,5)P2. The functional consequences of this event on signaling pathways are unknown. We show that the decrease in PI(4,5)P2 level under non-stimulated conditions inhibits PTEN activity leading to the aberrant activation of the oncoprotein Akt. This results in excessive cell proliferation and tumor progression in a xenograft mouse model. As well as defining a novel mechanism of Akt phosphorylation with important therapeutic consequences, we also demonstrate that differential expression levels of FGFR2, Plcγ1 and Grb2 correlate with patient survival. Oncogenesis through fluctuation in the expression levels of these proteins negates extracellular stimulation or mutation and defines them as novel prognostic markers in ovarian cancer.
Assuntos
Proteína Adaptadora GRB2/genética , Proteína Oncogênica v-akt/genética , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Fosfolipase C gama/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Feminino , Proteína Adaptadora GRB2/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias Ovarianas/patologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositóis/metabolismo , Fosfolipase C gama/biossíntese , Prognóstico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/biossíntese , Transdução de SinaisRESUMO
Primary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca(2+)-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca(2+)-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies.
Assuntos
Neoplasias Cardíacas/genética , Hemangiossarcoma/genética , Invasividade Neoplásica/genética , Fosfolipase C gama/genética , Apoptose/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Cardíacas/patologia , Hemangiossarcoma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Fosfolipase C gama/biossíntese , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Domínios de Homologia de src/genéticaRESUMO
The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Casitas B-lineage lymphoma (Cbl-b), Itch, gene related to anergy in lymphocyte, and deltex1 (DTX1). These E3 ligases attenuate T cell activation by targeting to signaling molecules. For example, Cbl-b and Itch promote the degradation of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in anergic Th1 cells. How these anergy-associated E3 ligases coordinate during T cell anergy remains largely unknown. In the current study, we found that PKCθ and PLC-γ1 are also downregulated by DTX1. DTX1 interacted with PKCθ and PLC-γ1 and stimulated the degradation of PKCθ and PLC-γ1. T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1(-/-) T cells, supporting the essential role of DTX1 in PKCθ downregulation. Similar to Cbl-b and Itch, DTX1 promoted monoubiquitination of PKCθ. Proteasome inhibitor did not inhibit DTX1-directed PKCθ degradation, but instead DTX1 directed the relocalization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated the possibility that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results suggest the coordination between E3 ligases during T cell anergy; DTX1 acts with Cbl-b to assure a more extensive silencing of PKCθ, whereas DTX1-mediated PKCθ degradation further stabilizes Cbl-b.
Assuntos
Proteínas de Ligação a DNA/genética , Isoenzimas/metabolismo , Proteína Oncogênica v-cbl/biossíntese , Proteína Quinase C/metabolismo , Proteólise , Células Th1/imunologia , Animais , Calcimicina/farmacologia , Linhagem Celular , Anergia Clonal , Proteínas de Ligação a DNA/biossíntese , Regulação para Baixo , Células HEK293 , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Células Jurkat , Ativação Linfocitária/imunologia , Lisossomos/imunologia , Camundongos , Camundongos Knockout , Proteína Oncogênica v-cbl/genética , Fosfolipase C gama/biossíntese , Fosfolipase C gama/metabolismo , Inibidores de Proteassoma/farmacologia , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Proteína Quinase C-theta , Interferência de RNA , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína-Tirosina Quinase ZAP-70/biossínteseRESUMO
BACKGROUND: Phospholipase C-γ1 (PLCγ1) is required for cellular migration during tumor progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to determine immunoexpression pattern of PLCγ1 in oral potentially malignant lesions (OPLs) and evaluate PLCγ1 usefulness as a biomarker for predicting clinical behavior in the carcinogenesis of OPL. METHODS: In a retrospective follow-up study, the expression pattern of PLCγ1 protein was determined using immunohistochemistry in samples from 68 patients, including untransformed cases (n = 38) and malignant-transformed cases (n = 30). The corresponding post-malignant lesions (OSCCs) were also performed. RESULTS: We observed that elevated expression of PLCγ1 in 40 of 68 (59%) general OPLs and 23 of 30 (77%) OSCCs compared with that in normal oral mucosa. Kaplan-Meier analysis revealed that patients with PLCγ1 positivity had a significantly higher incidence of OSCC than those with PLCγ1 negativity. Cox regression analysis revealed that PLCγ1 expression patterns were significantly associated with increased risk of malignant progression. In addition, the correlation between PLCγ1 expression in pre-malignant OPL and that in post-malignant OSCC was significant (P = 0.004). CONCLUSION: These data indicate that PLCγ1 expression in OPL correlated with oral cancer progression, and PLCγ1 may serve as a useful marker for the identification of high-risk OPL into OSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Bucais/metabolismo , Invasividade Neoplásica/patologia , Fosfolipase C gama/biossíntese , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Receptores ErbB/fisiologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/metabolismo , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Phospholipase Cγ1 (PLCγ1) is highly expressed in several tumors. We have previously reported that both stable and inducible PLCγ1 down-regulation resulted in an almost complete inhibition of breast cancer-derived experimental lung metastasis formation. The aim of our study is to evaluate the association between the expression of PLCγ1 and of PLCγ1 phosphorylated at Tyr1253 (PLCγ1-pY1253) and at Tyr783 (PLCγ1-pY783) with the clinical outcome of patients with node negative, T1/T2 breast cancers. The study groups consisted of 292 (training set) and 122 (validation set) patients presenting with primary unilateral breast carcinoma (T1-T2), with no evidence of nodal involvement and distant metastases. PLCγ1, PLCγ1-pY1253 and PLCγ1-pY783 protein expression were assessed by immunohistochemistry on tissue microarrays and the results correlated with the clinical data using Kaplan-Meier curves and multivariate Cox regression analysis. Tumor cells while expressing variable proportions of cytoplasmic PLCγ1, express PLCγ1-pY1253 and PLCγ1-pY783 predominantly in the nucleus. High expression of PLCγ1, and of its activated forms, is associated with a worse clinical outcome in terms of incidence of distant metastases, and not of local relapse in T1-T2, N0 breast cancer patients undergone adjuvant chemotherapy. PLCγ1 over-expression appears to be a reliable predictive surrogate marker of development of metastases. Thus, targeting PLCγ1 pathways might represent a potential therapeutic approach for the prevention of metastatic disease in breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Fosfolipase C gama/biossíntese , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fatores de RiscoRESUMO
Acquired drug resistance represents a major obstacle to using sunitinib for the treatment of solid tumors. Here, we examined the cellular and molecular alterations in tumors that are associated with acquired brain tumor resistance to sunitinib by using an in vivo model. U87MG tumors obtained from nude mice that received sunitinib (40 mg/kg/day) for 30 days were classified into sunitinib-sensitive and -resistant groups based on tumor volume and underwent targeted gene microarray and protein array analyses. The expression of several angiogenesis-associated genes was significantly modulated in sunitinib-treated tumors compared with those in control tumors (p<0.05), whereas no significant differences were observed between sunitinib-sensitive and -resistant tumors (p>0.05). Tumor vasculature based on microvessel density, neurogenin 2 chondroitin sulfate proteoglycan density, and α-smooth muscle actin density was also similar in sunitinib-treatment groups (p>0.05). The moderate increase in unbound sunitinib tumor-to-plasma area-under-the-curve ratio in sunitinib-resistant mice was accompanied by up-regulated ATP-binding cassette G2 expression in tumor. The most profound difference between the sunitinib-sensitive and -resistant groups was found in the expression of several phosphorylated proteins involved in intracellular signaling. In particular, phospholipase C-γ1 phosphorylation in sunitinib-resistant tumors was up-regulated by 2.6-fold compared with that in sunitinib-sensitive tumors (p<0.05). In conclusion, acquired sunitinib resistance in U87MG tumors is not associated with revascularization in tumors, but rather with the activation of alternate prosurvival pathways involved in an escape mechanism facilitating tumor growth and possibly insufficient drug uptake in tumor cells caused by an up-regulated membrane efflux transporter.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Glioma/tratamento farmacológico , Sobrevivência de Enxerto/efeitos dos fármacos , Indóis/farmacologia , Pirróis/farmacologia , Inibidores da Angiogênese/farmacocinética , Animais , Antineoplásicos/farmacocinética , Western Blotting , Imunofluorescência , Glioma/patologia , Humanos , Indóis/farmacocinética , Masculino , Camundongos , Camundongos Nus , Microdiálise , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fenótipo , Fosfolipase C gama/biossíntese , Fosfolipase C gama/genética , Reação em Cadeia da Polimerase , Pirróis/farmacocinética , SunitinibeRESUMO
This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-ß(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-ß(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-ß(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-ß(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the ß(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.
Assuntos
Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteoclastos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Animais , Reabsorção Óssea , Linhagem Celular , Integrina beta3/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Neuropeptídeos/biossíntese , Neuropeptídeos/metabolismo , Osteoclastos/fisiologia , Fosfolipase C gama/biossíntese , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-vav/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Quinase Syk , Proteínas rac de Ligação ao GTP/biossíntese , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Phospholipase C-γl (PLC-γl) expression is associated with cellular transformation. Notably, PLC-gamma is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-γl overexpression. We found that PLC-γl-transformed cells, but not vectortransformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-γl-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-γl-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-γl is highly up-regulated.
Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Transformação Celular Neoplásica , Enterotoxinas/toxicidade , Fibroblastos/efeitos dos fármacos , Mitose , Fosfolipase C gama/biossíntese , Animais , Células Cultivadas , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Fosfolipase C gama/genética , RatosRESUMO
Although endocrine therapy has dramatically improved the treatment of breast cancer therapeutic resistance and tumour recurrence occurs, even in estrogen receptor (ER) positive cases. Identifying and understanding the molecular mechanisms which underpin endocrine resistance is therefore important if future therapeutic strategies are to be developed. Members of the fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) families have been implicated in breast cancer development and progression. Our results demonstrate that culture of michigan cancer foundation - 1 (MCF)7 cells with FGF1 results in reduced sensitivity to tamoxifen in vitro. Furthermore, our tissue microarray expression data demonstrates that FGFR3 expression is increased in tamoxifen resistant breast tumours. To confirm that activation of FGFR3 reduced sensitivity to tamoxifen we used an inducible activation system and a constitutively active mutant of FGFR3 expressed in MCF7 cells. Activation of FGFR3 reduced sensitivity to tamoxifen and Fulvestrant but did not lead to phosphorylation of ER demonstrating that FGFR3 does not feedback to modulate ER activity. FGFR3 activation in MCF7 cells stimulated activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signalling pathways, both of which have been implicated in tamoxifen resistance in breast cancer. Furthermore, our data indicates that activation of phospholipase C gamma is a key-signalling event regulating MAPK and PI3K activation and that its activation reduces sensitivity to tamoxifen. Therefore, we hypothesise that FGFRs could play an integral part, not only in breast cancer development but also in resistance to endocrine-therapy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fulvestranto , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama/biossíntese , Fosfolipase C gama/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Tamoxifeno/farmacologiaRESUMO
The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.
Assuntos
Regulação da Expressão Gênica , Fosfolipase C gama/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Proteínas Quinases Ativadas por AMP , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
It is widely acknowledged that changes in intracellular calcium ion (Ca(2+)) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real-time PCR was used to analyse gene expression of 14 Ca(2+) signalling proteins in skin obtained from high tick-resistant (HR) and low tick-resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up-regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca(2+) signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca(2+) and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.
Assuntos
Sinalização do Cálcio/genética , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/parasitologia , Pele , Infestações por Carrapato/veterinária , Animais , Calsequestrina/biossíntese , Calsequestrina/genética , Bovinos , Doenças dos Bovinos/imunologia , Feminino , Interações Hospedeiro-Parasita , Imunidade Inata , Interleucina-2/biossíntese , Interleucina-2/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosfolipase C gama/biossíntese , Fosfolipase C gama/genética , Rhipicephalus/fisiologia , Pele/metabolismo , Pele/parasitologia , Infestações por Carrapato/imunologia , Infestações por Carrapato/metabolismo , Regulação para CimaRESUMO
Phospholipase C gamma-1 (PLCgamma1) phosphorylation is a key step in intracellular signal transduction in T cells. We used Hut-78 T lymphoma cells to demonstrate the effect of apicidin on cellular levels of the PLCgamma1 molecule. Using reverse-transcription, real-time quantitative PCR and Western blot analysis, we observed that apicidin reduced the PLCgamma1 transcript and protein contents in Hut-78 T lymphoma cells. Our results indicate that protein synthesis appears to be crucial in the apicidin decrease of PLCgamma1 mRNA steadiness. Moreover, we determined that apicidin reduces the half-life of PLCgamma1 mRNAs from approximately 2 to 4h. Since PLCgamma1 is considered a key molecule in signal transduction in T cells, apicidin may be useful in the treatment of some autoimmune diseases in which autoreactive T cells occur.
Assuntos
Inibidores de Histona Desacetilases , Peptídeos Cíclicos/farmacologia , Fosfolipase C gama/biossíntese , Transdução de Sinais/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Linfoma de Células T , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosforilação , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , CoelhosRESUMO
Cell motility and invasion play an essential role in the development of metastasis. Evidence suggests that the enzyme phospholipase Cgamma1 (PLCgamma1) may be involved in tumor progression and possibly development of metastasis. In this study, we show that down-regulation of PLCgamma1 expression severely impairs activation of the small GTP-binding protein Rac and cell invasion in breast cancer cell lines and U87 in vitro. Experimental metastasis assays in nude mice show that inducible knockdown of PLCgamma1 strongly inhibits development of MDA-MB-231-derived lung metastasis and reverts metastasis formation. In addition, analysis of 60 breast cancer patients' tissues revealed an increase of PLCgamma1 expression in metastasis compared with the primary tumor in 50% of tissues analyzed. These data show a critical role of PLCgamma1 in the metastatic potential of cancer cells, and they further indicate that PLCgamma1 inhibition has a therapeutic potential in the treatment of metastasis dissemination.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Fosfolipase C gama/biossíntese , Animais , Neoplasias da Mama/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Interferência de RNA , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We have identified a series of novel non-peptide compounds that activate the thrombopoietin-dependent cell line Ba/F3-huMPL. The compounds stimulated proliferation of Ba/F3-huMPL in the absence of other growth factors, but did not promote proliferation of the thrombopoietin-independent parent cell line Ba/F3. The thrombopoietin-mimetic compounds elicited signal-transduction responses comparable with recombinant human thrombopoietin, such as tyrosine phosphorylation of the thrombopoietin receptor, JAK (Janus kinase) 2, Tyk2 (tyrosine kinase 2), STAT (signal transducer and activator of transcription) 3, STAT5, MAPKs (mitogen-activated protein kinases), PLCgamma (phospholipase Cgamma), Grb2 (growth-factor-receptor-bound protein 2), Shc (Src homology and collagen homology), Vav, Cbl and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and increased the number of CD41(+) cells (megakaryocyte lineage) in cultures of human CD34(+) bone-marrow cells (haematopoietic stem cells). These findings suggest that this series of compounds are novel agonists of the human thrombopoietin receptor and are possible lead compounds for the generation of anti-thrombocytopaenia drugs.
Assuntos
Materiais Biomiméticos/farmacologia , Células da Medula Óssea/metabolismo , Receptores de Trombopoetina/agonistas , Transdução de Sinais/efeitos dos fármacos , Trombopoese/efeitos dos fármacos , Trombopoetina/farmacologia , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Proteína Adaptadora GRB2/biossíntese , Humanos , Camundongos , Fosfolipase C gama/biossíntese , Proteínas Quinases/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 11/biossíntese , Proteínas Proto-Oncogênicas c-cbl/biossíntese , Proteínas Proto-Oncogênicas c-vav/biossíntese , Receptores de Trombopoetina/metabolismo , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição STAT5/biossíntese , Proteínas Adaptadoras da Sinalização Shc/biossínteseRESUMO
Herbs have successfully been used in traditional Chinese medicine for centuries. However, their curative mechanisms remain largely unknown. In this study, we show that Wogonin, derived from the traditional Chinese medicine Huang-Qin (Scutellaria baicalensis Georgi), induces apoptosis in malignant T cells in vitro and suppresses growth of human T-cell leukemia xenografts in vivo. Importantly, Wogonin shows almost no toxicity on T lymphocytes from healthy donors. Wogonin induces prolonged activation of PLCgamma1 via H(2)O(2) signaling in malignant T cells, which leads to sustained elevation of cytosolic Ca(2+) in malignant but not normal T cells. Subsequently, a Ca(2+) overload leads to disruption of the mitochondrial membrane. The selective effect of Wogonin is due to its differential regulation of the redox status of malignant versus normal T cells. In addition, we show that the L-type voltage-dependent Ca(2+) channels are involved in the intracellular Ca(2+) mobilization in T cells. Furthermore, we show that malignant T cells possess elevated amounts of voltage-dependent Ca(2+) channels compared with normal T cells, which further enhance the cytotoxicity of Wogonin for malignant T cells. Taken together, our data show a therapeutic potential of Wogonin for the treatment of hematologic malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/farmacologia , Flavanonas/toxicidade , Leucemia de Células T/patologia , Fosfolipase C gama/biossíntese , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas , Indução Enzimática , Humanos , Células Jurkat/efeitos dos fármacos , Leucemia de Células T/tratamento farmacológico , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
OBJECTIVE: To study the effects of serum derived from rats treated with electroacupuncture at stomach meridian acupoints on the expressions of epidermal growth factor receptor (EGFR) signaling substances phospholipase C gamma-1 (PLC gamma-1), protein kinase C (PKC) and c-myc in gastric mucosal cells. METHODS: Sixty rats were randomly divided into normal group, stomach meridian group, gallbladder meridian group, stomach meridian plus PD153035 group and gallbladder meridian plus PD153035 group. Water-immersion and restrained stress methods were adopted for inducing gastric mucosal injury in the rats. Gastric mucosal cells were separated by using pronase digestion method, and incubated by PD153035, a EGFR inhibitor, and 100 ml/L serum. The expression of PLC gamma-1 in the gastric mucosal cells was tested by enzyme linked-immunosorbent assay (ELISA), while the expression of PKC by isotope incorporate assay and the expression of c-myc by reverse transcription polymerase chain reaction assay (RT-PCR). RESULTS: In gastric mucosal cells, weak expressions of PLC gamma-1, PKC and c-myc were seen in the normal group, and relatively strong expressions of PLC gamma-1, PKC and c-myc were seen in the stomach meridian group and the gallbladder meridian group, among which, the expressions of PLC gamma-1, PKC and c-myc in the stomach meridian group were the strongest, and there was a significant difference between the stomach meridian group and the gallbladder meridian group (P<0.01). Relative weak expressions of PLC gamma-1, PKC and c-myc were seen in the stomach meridian plus PD153035 group and the gallbladder meridian plus PD153035 group, and there was a significant difference between the stomach meridian group and the stomach meridian plus PD153035 group (P<0.01). CONCLUSIONS: The serum derived from the rats treated with electroacupuncture at stomach meridian acupoints can activate the EGFR singling pathway, and this provides an evidence for the theory of "relative particularity between meridians and viscera" in traditional Chinese medicine.