RESUMO
AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
Assuntos
Colestase , Endocitose , Estradiol , Hepatócitos , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Ácido Tauroquenodesoxicólico , Animais , Colestase/metabolismo , Colestase/induzido quimicamente , Colestase/prevenção & controle , Ratos , Transdução de Sinais/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/farmacologia , Estradiol/análogos & derivados , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Ácido Tauroquenodesoxicólico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Feminino , Masculino , Proteína Quinase C/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismoRESUMO
Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-ß-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2',7'-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Receptores ErbB , Estradiol , Óxido Nítrico , Fosfatidilinositol 3-Quinases , Fitoestrógenos , Transdução de Sinais , Vasodilatação , Animais , Fitoestrógenos/farmacologia , Estradiol/farmacologia , Óxido Nítrico/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores ErbB/metabolismo , Masculino , Isoflavonas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Genisteína/farmacologia , Receptores de Estrogênio/metabolismo , Ratos WistarRESUMO
Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.
Assuntos
Autofagia , Osteoclastos , Osteogênese , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ligante RANK , Serina-Treonina Quinases TOR , Ubiquinona , Animais , Camundongos , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
The aberrant activation of HER2 has a pivotal role in bone metastasis implantation and progression in several tumor types, including prostate cancer (PC). Trastuzumab and other anti-HER2 therapies, such as lapatinib, have been used in human breast cancer HER2 positive. Although HER2 overexpression has been reported in PC, anti-HER2 therapy response has revealed conflicting results. We investigated the potential of lapatinib in inhibiting cell migration and inducing apoptosis in two human (LNCaP and PC3) and two canine PC cell lines (PC1 and PC2). Cell migration and apoptosis were evaluated by Annexin V/PI analysis after lapatinib treatment. The transcriptome analysis of all cell lines before and after treatment with lapatinib was also performed. We found increased apoptosis and migration inhibition in LNCaP cells (androgen-sensitive cell line), while PC1, PC2, and PC3 cells showed no alterations after the treatment. The transcriptome analysis of LNCaP and PC3 cell lines showed 158 dysregulated transcripts in common, while PC1 and PC2 cell lines presented 82. At the doses of lapatinib used, we observed transcriptional modifications in all cell lines. PI3K/AKT/mTOR pathway were enriched in human PC cells, while canine PC cells showed enrichment of tyrosine kinase antitumor response and HER2-related pathways. In canine PC cells, the apoptosis failed after lapatinib treatment, possibly due to the downregulation of MAPK genes. Prostate cancer cells insensitive to androgens may be resistant to lapatinib through PI3K gene dysregulation. The association of lapatinib with PI3K inhibitors may provide a more effective antitumor response and clinical benefits to PC patients.
Assuntos
Antineoplásicos , Neoplasias da Mama , Neoplasias da Próstata , Masculino , Humanos , Animais , Cães , Lapatinib/farmacologia , Lapatinib/uso terapêutico , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Receptor ErbB-2/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Neoplasias da Mama/patologia , Apoptose , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos AntineoplásicosRESUMO
The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.
Assuntos
Neoplasias da Mama , Proteínas Sensoras de Cálcio Neuronal , Neuropeptídeos , Canal de Cátion TRPA1 , Feminino , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Proteínas Sensoras de Cálcio Neuronal/genética , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genéticaRESUMO
BACKGROUND: The objective of this research was to investigate how the combination of semen coicis extract and PD-1 inhibitors can potentially work together to enhance the anti-tumor effects, with a focus on understanding the underlying mechanism. METHODS: We obtained the active components and specific targets of semen coicis in the treatment of NSCLC from various databases, namely TCMSP, GeneCard, and OMIM. By utilizing the STRING database and Cytoscape software, we established a protein interaction network (PPI) for the active ingredient of semen coicis and the target genes related to NSCLC. To explore the potential pathways involved, we conducted gene ontology (GO) and biological pathway (KEGG) enrichment analyses, which were further supported by molecular docking technology. Additionally, we conducted cyto-inhibition experiments to verify the inhibitory effects of semen coicis alone or in combination with a PD-1 inhibitor on A549 cells, along with examining the associated pathways. Furthermore, we investigated the synergistic mechanism of these two drugs through cytokine release experiments and the PD-L1 expression study on A549 cells. RESULTS: Semen coicis contains two main active components, Omaine and (S)-4-Nonanolide. Its primary targets include PIK3R1, PIK3CD, PIK3CA, AKT2, and mTOR. Molecular docking experiments confirmed that these ingredients and targets form stable bonds. In vitro experiments showed that semen coicis demonstrates inhibitory effects against A549 cells, and this effect was further enhanced when combined with PD-1 inhibitors. PCR and WB analysis confirmed that the inhibition of the PI3K-AKT-mTOR pathway may contribute to this effect. Additionally, semen coicis was observed to decrease the levels of IFN-γ, IL-6, and TNF-α, promoting the recovery of the human anti-tumor immune response. And semen coicis could inhibit the induced expression of PDL1 of A549 cells stimulated by IFNγ as well. CONCLUSION: Semen coicis not only has the ability to kill tumor cells directly but also alleviates the immunosuppression found in the tumor microenvironment. Additionally, it collaboratively enhances the effectiveness of PD-1 inhibitors against tumors by blocking the activation of PI3K-AKT-mTOR.
Assuntos
Antineoplásicos , Coix , Neoplasias Pulmonares , Receptor de Morte Celular Programada 1 , Transdução de Sinais , Humanos , Células A549 , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Sinergismo Farmacológico , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Coix/química , Antineoplásicos/farmacologiaRESUMO
Interleukin 3 (IL-3) is a well-known pleiotropic cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells, triggering classical signaling pathways such as JAK/STAT, Ras/MAPK, and PI3K/Akt to carry out its functions. Interestingly, the IL-3 receptor is also expressed in non-hematopoietic cells, playing a crucial role in cell survival. Our previous research demonstrated the expression of the IL-3 receptor in neuron cells and its protective role in neurodegeneration. Glutamate, a principal neurotransmitter in the central nervous system, can induce cellular stress and lead to neurotoxicity when its extracellular concentrations surpass normal levels. This excessive glutamate presence is frequently observed in various neurological diseases. In this study, we uncover the protective role of IL-3 as an inhibitor of glutamate-induced cell death, analyzing the cytokine's signaling pathways during its protective effect. Specifically, we examined the relevance of JAK/STAT, Ras/MAPK, and PI3 K signaling pathways in the molecular mechanism triggered by IL-3. Our results show that the inhibition of JAK, ERK, and PI3 K signaling pathways, using pharmacological inhibitors, effectively blocked IL-3's protective role against glutamate-induced cell death. Additionally, our findings suggest that Bcl-2 and Bax proteins may be involved in the molecular mechanism triggered by IL-3. Our investigation into IL-3's ability to protect neuronal cells from glutamate-induced damage offers a promising therapeutic avenue with potential clinical implications for several neurological diseases characterized by glutamate neurotoxicity.
Assuntos
Interleucina-3 , Neuroblastoma , Humanos , Ácido Glutâmico/toxicidade , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Interleucina-3 , Linhagem Celular , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Açaí seed extract (ASE) is obtained from Euterpe oleracea Mart. (açaí) plant (Amazon region) has high nutritional and functional value. ASE is rich in polyphenolic compounds, mainly proanthocyanidins. Proanthocyanidins can modulate the immune system and oxidative stress by inhibiting the toll-like receptor-4 (TLR-4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) pathway. A great deal of evidence suggests that inflammatory cytokines and oxidative stress contribute to the pathogenesis of intestinal mucositis, and these events can lead to intestinal dysmotility. We hypothesized that ASE acts as an anti-inflammatory and antioxidant compound in intestinal mucositis induced by 5-fluorouracil (5-FU) through modulation of the TLR-4/MyD88/phosphatidylinositol-3-kinase α/mechanistic target of rapamycin/NF-κBp65 pathway. The animals were divided into linear 5-FU (450 mg/kg) and 5-FU + ASE (10, 30, and 100 mg/kg) groups. The weight loss of the animals was evaluated daily. Samples from duodenum, jejunum, and ileum were obtained for histopathological, biochemical, and functional analyses. ASE reduced weight loss, inflammatory parameters (interleukin-1ß; tumor necrosis factor-α; myeloperoxidase activity) and the gene expression of mediators involved in the TLR-2/MyD88/NF-κB pathway. ASE prevented histopathological changes with beneficial effects on gastrointestinal transit delay, gastric emptying, and intestinal absorption/permeability. In conclusion, ASE protects the integrity of the intestinal epithelial barrier by inhibiting the TLR/MyD88/PI3K/mechanistic target of rapamycin/NF-κBp65 pathway.
Assuntos
Euterpe , Fluoruracila , Mucosite , Fator 88 de Diferenciação Mieloide , Extratos Vegetais , Polifenóis , Sementes , Transdução de Sinais , Serina-Treonina Quinases TOR , Receptor 4 Toll-Like , Animais , Receptor 4 Toll-Like/metabolismo , Mucosite/induzido quimicamente , Mucosite/tratamento farmacológico , Mucosite/prevenção & controle , Mucosite/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sementes/química , Polifenóis/farmacologia , Masculino , Euterpe/química , Camundongos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fator de Transcrição RelA/metabolismo , Antioxidantes/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , NF-kappa B/metabolismoRESUMO
The incidence of non-alcoholic fatty liver (NAFLD) remains high, and many NAFLD patients suffer from severe ischemia-reperfusion injury (IRI). Currently, no practical approach can be used to treat IRI. Puerarin plays a vital role in treating multiple diseases, such as NAFLD, stroke, diabetes, and high blood pressure. However, its role in the IRI of the fatty liver is still unclear. We aimed to explore whether puerarin could protect the fatty liver from IRI. C57BL/6J mice were fed with a high-fat diet (HFD) followed by ischemia reperfusion injury. We showed that hepatic IRI was more severe in the fatty liver compared with the normal liver, and puerarin could significantly protect the fatty liver against IRI and alleviate oxidative stress. The PI3K-AKT signaling pathway was activated during IRI, while liver steatosis decreased the level of activation. Puerarin significantly protected the fatty liver from IRI by reactivating the PI3K-AKT signaling pathway. However, LY294002, a PI3K-AKT inhibitor, attenuated the protective effect of puerarin. In conclusion, puerarin could significantly protect the fatty liver against IRI by activating the PI3K-AKT signaling pathway.
Assuntos
Isoflavonas , Hepatopatia Gordurosa não Alcoólica , Traumatismo por Reperfusão , Animais , Camundongos , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fígado/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/metabolismoRESUMO
Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.