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Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.
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Arginina , Ferroptose , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Animais , Arginina/metabolismo , Arginina/análogos & derivados , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Camundongos , Linhagem Celular Tumoral , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Retroalimentação Fisiológica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , Camundongos Nus , Transdução de Sinais , Separação de Fases , Proteínas de Ligação a RNARESUMO
Circulating tumor cells (CTCs) are considered an important factor involved in tumor metastasis and can overcome mechanical interactions to gain the ability to distant metastasis. The previous study had shown that the suspension state could regulate the stemness of breast cancer cells (BCCs). However, the specific molecular mechanisms involved have not yet been explored clearly. In this study, MCF-7 and MDA-MBA-231 BCCs were cultured in suspension and adherent. The effect of suspension state on BCCs was further elucidated by observing suspension cell clusters, sorting CD44+/CD24- cell subpopulation and detecting self-renewal ability. Furthermore, it was found that glycogen synthase kinase-3ß (GSK-3ß) was significantly down-regulated in MCF-7 suspension cells along with the activation of the Wnt/ß-catenin signaling, but the converse was true for MDA-MB-231 cells. Subsequently, GSK-3ß was differentially expressed in MCF-7 suspension cells. The activation of the Wnt/ß-catenin signaling, epithelial-mesenchymal transition (EMT) and stemness were all inhibited when GSK-3 was overexpressed in suspension MCF-7 cells. While GSK-3ß was down-regulated, it further promoted the Wnt/ß-catenin signaling, mesenchymal characteristic and stemness of MCF-7 cells. This study demonstrated that suspension state could activate the Wnt/ß-catenin signaling by inhibiting GSK-3ß to promote the stemness of epithelial BCCs, providing a therapeutic strategy for targeted CTCs.
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Neoplasias da Mama , beta Catenina , Feminino , Humanos , beta Catenina/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Quinase 3 da Glicogênio Sintase , Glicogênio Sintase Quinase 3 beta , Via de Sinalização WntRESUMO
Background: Ciprofol is currently used for painless gastrointestinal endoscopy and anesthesia induction. However, whether it is superior to propofol and its optimal dose remains unknown. Methods: A total of 149 patients, 63 males and 86 females, aged 18-80 years, BMI 18-28 kg/m2, ASA I-III, were divided randomly into four groups: propofol group (group P, n = 44), ciprofol 0.2mg/kg group (group C2, n = 38), ciprofol 0.3mg/kg group (group C3, n = 36) and ciprofol 0.4 mg/kg group (group C4, n = 31). Groups C2, C3 and C4 had injected IV with ciprofol 0.2, 0.3 and 0.4 mg/kg, respectively. Group P had injected IV with propofol 1.5mg/kg. The time for disappearance of the eyelash reflex, gastrointestinal endoscopy time, recovery time, and the Modified Observer's Assessment of Alertness/Sedation (MOAA/S) score at awakening (T1), 15 minutes after awakening (T2) and 30 minutes after awakening (T3) were recorded. Results: Compared with group P, the time to fall asleep was significantly shortened, and the incidence of nausea and vomiting and injection pain was significantly lower in groups C2, C3 and C4 (P < 0.05). There was no significant difference in recovery time and recovery quality between each group (P > 0.05). Compared with group P and C4, the incidence of hypotension and respiratory depression was significantly lower in groups C2 and C3 (P < 0.05). Conclusion: The appropriate dose of ciprofol for painless gastrointestinal endoscopy is more advantageous than propofol in hemodynamics and respiratory stability, with less injection pain and nausea and vomiting, which is worthy of clinical promotion.
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Propofol , Masculino , Feminino , Humanos , Endoscopia Gastrointestinal , Dor/tratamento farmacológico , Dor/induzido quimicamente , Hemodinâmica , Anestesia GeralRESUMO
PURPOSE: Pathological complete response(pCR) during neoadjuvant chemotherapy(NAC) has been proposed as a predictor for better prognosis in breast cancer. However, few studies compare the outcomes of patients receiving NAC and adjuvant chemotherapy(AC). METHODS: We retrospectively matched the patients who received NAC(N = 462) and AC(N = 462) by age, time of diagnosis, and primary clinical stage using the propensity score match in breast cancer patients treated in Sir Run Run Shaw Hospital with the median follow up of 67 months. Death from breast cancer and recurrence were used as endpoints. A multivariable Cox models were used to estimate the hazard ratios for breast-cancer specific survival (BCSS) and DFS. A multivariable logistic regression model was simulated to predict pCR. RESULTS: In patients who received NAC, 18.0%(83/462) patients achieved pCR, while the rest of the patients did not. pCR subgroup demonstrated significant better BCSS and DFS than patients receiving AC(BCSS: HR = 0.39, 95% CI:0.12-0.93, P = 0.03; DFS: HR = 0.16, 95%CI 0.009-0.73, P = 0.013) and non-pCR patients(BCSS: HR = 0.32, 95%CI 0.10-0.77, P = 0.008; DFS: HR = 0.12, 95%CI 0.007-0.55, P = 0.002). Patients who received AC demonstrated insignificant survival compared to non-pCR patients(BCSS: HR= 0.82, 95%CI 0.62-1.10, P = 0.19; DFS: HR = 0.75, 95%CI 0.53-1.07, P = 0.12). Patients with AC had significant better DFS than non-pCR patients(HR = 0.33, 95% CI 0.10-0.94, P = 0.04) in luminal B Her2+ patients. More NAC cycles(>2), TNBC, lower cT stage, and mixed histology indicate higher possibility of pCR(AUC = 0.89). CONCLUSION: pCR patients with NAC indicated better prognosis than patients receiving AC or non-pCR patients from NAC. The timing of chemotherapy may need carefully pondering in luminal B Her2+ patients.
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Neoplasias da Mama , Humanos , Pré-Escolar , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Terapia Neoadjuvante , Estudos Retrospectivos , Prognóstico , Quimioterapia AdjuvanteRESUMO
The tumor microenvironment is proposed to contribute substantially to the progression of cancers, including breast cancer. Cancer-associated fibroblasts (CAFs) are the most abundant components of the tumor microenvironment. Studies have revealed that CAFs in breast cancer originate from several types of cells and promote breast cancer malignancy by secreting factors, generating exosomes, releasing nutrients, reshaping the extracellular matrix, and suppressing the function of immune cells. CAFs are also becoming therapeutic targets for breast cancer due to their specific distribution in tumors and their unique biomarkers. Agents interrupting the effect of CAFs on surrounding cells have been developed and applied in clinical trials. Here, we reviewed studies examining the heterogeneity of CAFs in breast cancer and expression patterns of CAF markers in different subtypes of breast cancer. We hope that summarizing CAF-related studies from a historical perspective will help to accelerate the development of CAF-targeted therapeutic strategies for breast cancer.
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Neoplasias da Mama , Fibroblastos Associados a Câncer , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Feminino , Humanos , Microambiente TumoralRESUMO
[This corrects the article DOI: 10.3389/fonc.2021.697950.].
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Ferroptosis, which is characterized by intracellular iron accumulation and lipid peroxidation, is a newly described form of regulated cell death that may play a key role in tumour suppression. In the present study, we investigated the expression profiles and biological effects of fascin actin-bundling protein 1 (Fascin, gene name FSCN1) in breast cancer. In addition, bioinformatics analysis of the TCGA cancer database and gain- and loss-of-function studies showed that Fascin enhances sensitivity to erastin-induced ferroptosis. Mechanistically, Fascin directly interacts with cysteine/glutamate transporter (xCT, gene name SLC7A11) and decreases its stability via the ubiquitin-mediated proteasome degradation pathway. Furthermore, we observed that Fascin is substantially upregulated in tamoxifen-resistant breast cancer cell lines, and drug-resistant cells were also more vulnerable to erastin-induced ferroptosis. Taken together, our findings reveal a previously unidentified role of Fascin in ferroptosis by regulating xCT. Thus, ferroptosis activation in breast cancer with high Fascin level may serve as a potential treatment.
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Neoplasias da Mama , Proteínas de Transporte , Ferroptose , Proteínas dos Microfilamentos , Piperazinas , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Ferroptose/genética , Humanos , Proteínas dos Microfilamentos/genética , Piperazinas/farmacologiaRESUMO
Aberrant methylation has been regarded as a hallmark of cancer. 5-hydroxymethylcytosine (5hmC) is recently identified as the ten-eleven translocase (ten-eleven translocase)-mediated oxidized form of 5-methylcytosine, which plays a substantial role in DNA demethylation. Cell-free DNA has been introduced as a promising tool in the liquid biopsy of cancer. There are increasing evidence indicating that 5hmC in cell-free DNA play an active role during carcinogenesis. However, it remains unclear whether 5hmC could surpass classical markers in cancer detection, treatment, and prognosis. Here, we systematically reviewed the recent advances in the clinic and basic research of DNA 5-hydroxymethylation in cancer, especially in cell-free DNA. We further discuss the mechanisms underlying aberrant 5hmC patterns and carcinogenesis. Synergistically, 5-hydroxymethylation may act as a promising biomarker, unleashing great potential in early cancer detection, prognosis, and therapeutic strategies in precision oncology.
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Chemoresistance is a daunting challenge to the prognosis of patients with breast cancer. Signal transducer and activator of transcription (STAT) 5a plays vital roles in the development of various cancers, but its function in breast cancer is controversial, and its role in chemoresistance in breast cancer remains unexplored. Here we identified STAT5a as a chemoresistance inducer that regulates the expression of ABCB1 in breast cancer and can be targeted by pimozide, an FDA-approved psychotropic drug. First, we found that STAT5a and ABCB1 were expressed at higher levels in doxorubicin-resistant cell lines and chemoresistant patients, and their expression was positively correlated. Then, we confirmed the essential roles of STAT5a and ABCB1 in doxorubicin resistance in breast cancer cells and the regulation of ABCB1 transcription by STAT5a. Subsequently, the efficacy of pimozide in inhibiting STAT5a and sensitizing doxorubicin-resistant breast cancer cells was tested. Finally, we verified the role of STAT5a in doxorubicin resistance in breast cancer and the efficacy of pimozide in reversing this resistance in vivo. Our study demonstrated the vital role of STAT5a in doxorubicin resistance in breast cancer. Targeting STAT5a might be a promising strategy for treating doxorubicin-resistant breast cancer. Moreover, repurposing pimozide for doxorubicin resensitization is attractive due to the safety profile of pimozide.
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BACKGROUND: Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid peroxidation and is involved in various pathophysiological conditions, including cancer. Targeting ferroptosis is considered to be a novel anti-cancer strategy. The identification of FDA-approved drugs as ferroptosis inducers is proposed to be a new promising approach for cancer treatment. Despite a growing body of evidence indicating the potential efficacy of the anti-diabetic metformin as an anti-cancer agent, the exact mechanism underlying this efficacy has not yet been fully elucidated. METHODS: The UFMylation of SLC7A11 is detected by immunoprecipitation and the expression of UFM1 and SLC7A11 in tumor tissues was detected by immunohistochemical staining. The level of ferroptosis is determined by the level of free iron, total/lipid Ros and GSH in the cells and the morphological changes of mitochondria are observed by transmission electron microscope. The mechanism in vivo was verified by in situ implantation tumor model in nude mice. RESULTS: Metformin induces ferroptosis in an AMPK-independent manner to suppress tumor growth. Mechanistically, we demonstrate that metformin increases the intracellular Fe2+ and lipid ROS levels. Specifically, metformin reduces the protein stability of SLC7A11, which is a critical ferroptosis regulator, by inhibiting its UFMylation process. Furthermore, metformin combined with sulfasalazine, the system xc- inhibitor, can work in a synergistic manner to induce ferroptosis and inhibit the proliferation of breast cancer cells. CONCLUSIONS: This study is the first to demonstrate that the ability of metformin to induce ferroptosis may be a novel mechanism underlying its anti-cancer effect. In addition, we identified SLC7A11 as a new UFMylation substrate and found that targeting the UFM1/SLC7A11 pathway could be a promising cancer treatment strategy.
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Sistema y+ de Transporte de Aminoácidos/metabolismo , Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Metformina/administração & dosagem , Proteínas/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Ferroptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Células MCF-7 , Metformina/farmacologia , Metilação , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adriamycin (ADM) is currently one of the most effective chemotherapeutic agents in breast cancer treatment. However, growing resistance to ADM could lead to treatment failure and poor outcome. PLAC8 was reported as a novel highly conserved protein and functioned as an oncogene or tumour suppressor in various tumours. Here, we found higher PLAC8 expression was correlated with worse outcome and aggressive phenotype in breast cancer. Breast cancer patients with higher PLAC8 expression showed potential ADM resistance. In vitro experiments further confirmed that PLAC8 inhibited by siRNA or enforced overexpression by infecting pcDNA3.1(C)-PLAC8 plasmid correspondingly decreased or increased ADM resistance. Subsequently, we demonstrated that ectopic PLAC8 expression in MCF-7/ADMR cell blocked the accumulation of the autophagy-associated protein LC3 and resulted in cellular accumulation of p62. Rapamycin-triggered autophagy significantly increased cell response to ADM, while the autophagy inhibitor 3-MA enhanced ADM resistance. 3-MA and PLAC8 could synergistically cause ADM resistance via blocking the autophagy process. Additionally, the down-regulation of p62 by siRNA attenuated the activation of autophagy and PLAC8 expression in breast cancer cells. Thus, our findings suggest that PLAC8, through the participation of p62, inhibits autophagy and consequently results in ADM resistance in breast cancer. PLAC8/p62 pathway may act as novel therapeutic targets in breast cancer treatment and has potential clinical application in overcoming ADM resistance.
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Autofagia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Mamárias Experimentais/metabolismo , Proteínas/metabolismo , Animais , Antibióticos Antineoplásicos/uso terapêutico , Antibióticos Antineoplásicos/toxicidade , Doxorrubicina/uso terapêutico , Doxorrubicina/toxicidade , Feminino , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Nus , Proteínas/genéticaRESUMO
Drug resistance is a daunting challenge in the treatment of breast cancer, making it an urgent problem to solve in studies. Cell lines are important tools in basic and preclinical studies; however, few breast cell lines from drug-resistant patients are available. Herein, we established a novel HER2-positive breast cancer cell line from the pleural effusion of a drug-resistant metastatic breast cancer patient. This cell line has potent proliferative capability and tumorigenicity in nude mice but weak invasive and colony-forming capability. The molecular subtype of the cell line and its sensitivity to chemotherapeutics and HER2-targeting agents are different from those of its origin, suggesting that the phenotype changes between the primary and metastatic forms of breast cancer.
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OBJECTIVES: The mechanical forces on circulating tumor cells (CTCs) should not be ignored in blood and it is more essential that CTCs can overcome and utilize the mechanical interaction to acquire the ability of distant metastasis. At present there are few studies on how suspension mechanics regulates the behavior of tumor cells. The aim of the study was to explore the effects of suspension state on the epithelial-mesenchymal transition (EMT) and stemness of breast CTCs and the molecular mechanisms involved. RESULTS: Suspension state could regulate the program of EMT in breast cancer cells, which supported the complex dynamic concept of EMT. It is that the Ras homolog family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway was activated by suspension state in MCF-7 cells instead of MDA-MB-231 cells. In addition, suspension state increased the stemness of breast cancer cells from different aspects. CONCLUSION: The study highlighted the emergence of hybrid epithelial/mesenchymal (E/M) state during hematogenous metastasis and the plasticity of CTCs caused by cancer stem cells, further providing novel insights into clinical monitoring of CTCs and therapeutic strategies.
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Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular/fisiologia , Feminino , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/fisiologia , FenótipoRESUMO
Adjuvant chemotherapy(AC) plays a substantial role in the treatment of locally advanced gastric cancer (LAGC), but the response remains poor. We aims to improve its efficacy in LAGC. Therefore, we identified the expression of eight genes closely associated with platinum and fluorouracil metabolism (RRM1, RRM2, RRM2B, POLH, DUT, TYMS, TYMP, MKI67) in the discovery cohort (N=291). And we further validated the findings in TCGA (N=279) and GEO. Overall survival (OS) was used as an endpoint. Univariate and multivariate Cox models were applied. A multivariate Cox regression model was simulated to predict the OS. In the discovery cohort, the univariate Cox model indicated that AC was beneficial to high-RRM1, high-DUT, low-RRM2, low-RRM2B, low-POLH, low-KI67, low-TYMS or low-TYMP patients, the results were validated in the TCGA cohort. The multivariate Cox model showed consistent results. Cumulative analysis indicated that patients with low C-Score respond poorly to the AC, whereas the high and medium C-Score patients significantly benefit from AC. A risk model based on the above variables successfully predicted the OS in both cohorts (AUC=0.75 and 0.67, respectively). Further validation in a panel of gastric cancer cell (GC) lines (N=37) indicated that C-Score is significantly associated with IC50 value to fluorouracil. Mutation profiling showed that C-Score was associated with the number and types of mutations. In conclusion, we successfully simulated a predictive signature for the efficacy of AC in LAGC patients and further explored the potential mechanisms. Our findings could promote precision medicine and improve the prognosis of LAGC patients.
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Ferroptosis is a recently discovered distinct type of regulated cell death caused by the accumulation of lipid-based ROS. Metabolism and expression of specific genes affect the occurrence of ferroptosis, making it a promising therapeutic target to manage cancer. Here, we describe the current status of ferroptosis studies in breast cancer and trace the key regulators of ferroptosis back to previous studies. We also compare ferroptosis to common regulated cell death patterns and discuss the sensitivity to ferroptosis in different subtypes of breast cancer. We propose that viewing ferroptosis-related studies from a historical angle will accelerate the development of ferroptosis-based biomarkers and therapeutic strategies in breast cancer.
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The cysteine-rich lysosomal protein placenta-specific 8 (PLAC8), also called onzin, has been shown to be involved in many types of cancers, and its role is highly dependent on cellular and physiological contexts. However, the precise function of PLAC8 in breast cancer (BC) progression remains unclear. In this study, we investigated both the clinical significance and biological functions of PLAC8 in BC progression. First, high PLAC8 expression was observed in primary BC tissues compared with adjacent normal tissues through immunohistochemistry analysis. The results of in vitro and in vivo assays further confirmed that PLAC8 overexpression promotes cell proliferation and suppress BC cell apoptosis, whereas PLAC8 silencing has the opposite effect. In addition, the forced expression of PLAC8 greatly induces cell migration, partially by affecting the EMT-related genes, including down-regulating E-cadherin expression and facilitating vimentin expression. Further mechanistic analysis confirmed that PLAC8 contributes to cell proliferation and suppresses cell apoptosis in BC by activating the PI3K/AKT/NF-κB pathway. The results of our study provide new insights into an oncogenic role of PLAC8 and reveal a novel PLAC8/ PI3K/AKT/NF-κB pathway as a potential therapeutic target for BC.
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Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Neoplasias da Mama/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND: Protease-activated receptors (PARs) can be stimulated by thrombin and other proteases generated by periodontal pathogens. Activation of PARs in gingival fibroblasts (GFs) can modulate wound healing and inflammatory responses in gingival tissues. METHODS: The mRNA expression of PARs and early responsive genes in GFs and other oral cells was studied by reverse transcription-polymerase chain reaction. Western blotting was used to study the activation of p38 and cAMP responsive element binding protein (CREB)/activating transcription factor (ATF)-1 as well as Ras. RESULTS: GFs, dental pulp cells, and buccal mucosal fibroblasts expressed PAR-1 and -3 receptors, whereas gingival keratinocytes expressed PAR-1 and -2 receptors. Stimulation of GFs by thrombin rapidly activated Ras signaling and the phosphorylation of CREB/ATF-1 and p38. Thrombin also stimulated the expression of c-fos in GFs within 1 hour of exposure. Stimulation of c-jun mRNA expression showed biphasic responses with two peaks after 1 and 8 hours of exposure. Elevated c-myc expression in GFs by thrombin was noted after 2 hours of exposure. Moreover, the stimulation of c-fos and c-myc mRNA expression by thrombin can be attenuated by D-Phe-Pro-ArgCH(2)Cl, a serine-proteinase inhibitor. CONCLUSIONS: PAR activation during gingival wounding or inflammation may stimulate Ras-CREB/ATF-1 signaling and c-fos, c-jun, and c-myc expression. This might be due to the proteinase activity of thrombin. These signaling events are important for wound healing and inflammatory responses in gingival tissues.
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Fator 1 Ativador da Transcrição/efeitos dos fármacos , Proteína de Ligação a CREB/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Western Blotting , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Fibroblastos/citologia , Gengiva/citologia , Humanos , Queratinócitos/efeitos dos fármacos , Mucosa Bucal/citologia , Mucosa Bucal/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptor PAR-1/efeitos dos fármacos , Receptor PAR-2/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores de Serina Proteinase/farmacologia , Trombina/antagonistas & inibidores , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Transforming growth factor beta (TGF-beta) may regulate the biological activities of dental pulp cells. We found that human dental pulp cells expressed TGF-beta1, TGF-beta2, and a little amount of TGF-beta3 messenger RNA (mRNA). The exposure of pulp cells to TGF-beta2 induced the phosphorylation of Smad2/3, Smad1/5/8, and extracellular regulated-kinase 1/2 (ERK1/2) as observed by Western blotting. Exposure to TGF-beta2 decreased the alkaline phosphatase (ALP) mRNA expression and enzyme activity. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-beta ALK-4, ALK-5, and ALK-7 receptors) but not U0126 (a MEK1 inhibitor) prevented the inhibition of viable cell number, ALP activity, and mRNA expression by TGF-beta2 in dental pulp cells. These results suggest that TGF-beta may affect the growth and differentiation of dental pulp cells via an autocrine fashion by activation of the ALK/Smad2/3-signal transduction pathways. TGF-beta2 possibly regulates the differentiation of pulp cell at specific stages synergistically with other factors.
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Polpa Dentária/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad Reguladas por Receptor/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Fosfatase Alcalina/metabolismo , Benzamidas/farmacologia , Diferenciação Celular , Proliferação de Células , Polpa Dentária/citologia , Dioxóis/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , MAP Quinase Quinase Quinases/fisiologia , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta2/biossínteseRESUMO
Sanguinarine is a benzopheanthridine alkaloid present in the root of Sanguinaria canadensis L. and Chellidonium majus L. In this study, sanguinarine (2 and 3 microM) exhibited cytotoxicity to KB cancer cells by decreasing MTT reduction to 83% and 52% of control after 24-h of exposure. Sanguinarine also inhibited the colony forming capacity (>52-58%) and growth of KB cancer cells at concentrations higher than 0.5-1 microM. Short-term exposure to sanguinarine (>0.5 microM) effectively suppressed the adhesion of KB cells to collagen and fibronectin (FN). Sanguinarine (2 and 3 microM) induced evident apoptosis as indicated by an increase in sub-G0/G1 populations, which was detected after 6-h of exposure. Only a slight increase in cells arresting in S-phase and G2/M was noted. Induction of KB cell apoptosis and necrosis by sanguinarine (2 and 3 microM) was further confirmed by Annexin V-PI dual staining flow cytometry and the presence of DNA fragmentation. The cytotoxicity by sanguinarine was accompanied by an increase in production of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential as indicated by single cell flow cytometric analysis of DCF and rhodamine fluorescence. NAC (1 and 3 mM) and catalase (2000 U/ml) prevented the sanguinarine-induced ROS production and cytotoxicity, whereas dimethylthiourea (DMT) showed no marked preventive effect. These results suggest that sanguinarine has anticarcinogenic properties with induction of ROS production and mitochondrial membrane depolarization, which mediate cancer cell death.