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Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system with a high mortality rate. The upregulation of RING finger protein 135 (RNF135), an E3 ligase, has been observed in GBM, but the associated mechanisms have not been fully elucidated. The aim of the present study was to identify the substrate of RNF135 and study its functions in GBM. Bioinformatics analyses were performed. In addition, RNF135 was overexpressed or knocked down in human U87 and U251 GBM cells, and the effect on cell proliferation was analyzed using Cell Counting Kit-8 and colony formation assays. Furthermore, the interaction of RNF135 with its potential substrate was analyzed using glutathione S-transferase, yeast two-hybrid, immunoprecipitation (IP), co-IP and immunoblotting assays. Bioinformatics analysis indicated that RNF135 serves as a marker of poor prognosis in GBM. The overexpression of RNF135 was demonstrated to promote the proliferation of GBM cells, while the knockdown of RNF135 inhibited GBM cell growth. In addition, the results of the interaction experiments indicate that RNF135 interacts with p21 and mediates the degradation of p21 by ubiquitination. The major site of RNF135-mediated p21 ubiquitination was identified as K163. Further experiments demonstrated that RNF135 promotes the proliferation of GBM cells mainly via p21. In summary, these findings suggest that RNF135 has potential as a therapeutic target for the treatment of GBM.
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Background: At a crucial time with the rapid spread of Omicron severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus variant globally, we conducted a study to evaluate the efficacy and safety of arbidol tablets in the treatment of this variant. Methods: From Mar 26 to April 26, 2022, we conducted a prospective, open-labeled, controlled, and investigator-initiated trial involving adult patients with confirmed Omicron variant infection. Patients with asymptomatic or mild clinical status were stratified 1:2 to receive either standard-of-care (SOC) or SOC plus arbidol tablets (oral administration of 200 mg per time, three times a day for 5 days). The primary endpoint was the negative conversion rate within the first week. Results: A total of 367 patients were enrolled in the study; 246 received arbidol tablet treatment, and 121 were in the control group. The negative conversion rate of SARS-CoV-2 within the first week in patients receiving arbidol tablets was significantly higher than that of the SOC group [47.2% (116/246) vs. 35.5% (43/121); odds ratio (OR), 1.619; 95% confidence interval (CI): 1.034-2.535; P=0.035]. Compared to those in the SOC group, patients receiving arbidol tablets had a shorter negative conversion time [median 8.3 vs. 10.0 days; hazard ratio (HR), 0.645; 95% CI: 0.516-0.808; P<0.001], and a shorter duration of hospitalization (median 11.4 vs. 13.7 days; HR, 1.214; 95% CI: 0.966-1.526; P<0.001). Moreover, the addition of arbidol tablets led to better recovery of declined blood lymphocytes, CD3+, CD4+, and CD8+ cell counts. The most common adverse event (AE) was transaminase elevation in patients treated with arbidol tablets (3/246, 1.2%). No one withdrew from the study due to AEs or disease progression. Conclusions: As a whole, arbidol may represent an effective and safe treatment in asymptomatic-mild patients suffering from Omicron variant during the pandemic of coronavirus disease 2019 (COVID-19).
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PURPOSE: The dopamine agonists (DA) have been used widely to treat prolactinomas. However, it is difficult to predict whether the patient will be responsive to DA treatment. METHODS: We aimed to investigate whether the in vivo expression of DRD2 based on 18F-fallypride PET/MR could predict the therapeutic effect of DA on prolactinomas. Seven patients with prolactinomas completed 18F-fallypride PET/MR. Among them, three patients underwent surgery and further tumor immunohistochemistry. Imaging findings and immunohistochemical staining were compared with treatment outcomes. RESULTS: 18F-fallypride PET/MR was visually positive in 7 of 7 patients, and DRD2 target specificity could be confirmed by immunohistochemical staining. A significantly lower tracer standard uptake value (SUV) could be detected in the resistant patients (n = 3) than in the sensitive patients (n = 4; SUVmean, 4.67 ± 1.32 vs. 13.57 ± 2.42, p < 0.05). DRD2 expression determined by 18F-fallypride PET/MR corresponded with the DA treatment response. CONCLUSION: 18F-fallypride PET/MR may be a promising technique for predicting DA response in patients with prolactinoma.
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Neoplasias Hipofisárias , Prolactinoma , Humanos , Prolactinoma/diagnóstico por imagem , Prolactinoma/tratamento farmacológico , Agonistas de Dopamina/uso terapêutico , Projetos Piloto , Receptores de Dopamina D2/metabolismo , Neoplasias Hipofisárias/diagnóstico por imagem , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/metabolismo , Tomografia por Emissão de PósitronsRESUMO
BACKGROUND: Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors that are classified into seven histological subtypes, including lactotroph, somatotroph, corticotroph, thyrotroph, gonadotroph, null cell, and plurihormonal PitNETs. However, the molecular characteristics of these types of PitNETs are not completely clear. METHODS: A total of 180 consecutive cases of PitNETs were collected to perform RNA sequencing. All subtypes of PitNETs were distinguished by unsupervised clustering analysis. We investigated the regulation of TPIT by TRIM65 and its effects on ACTH production and secretion in ACTH-secreting pituitary cell lines, as well as in murine models using biochemical analyses, confocal microscopy, and luciferase reporter assays. RESULTS: A novel subtype of PitNETs derived from TPIT lineage cells was identified as with normal TPIT transcription but with lowered protein expression. Furthermore, for the first time, TRIM65 was identified as the E3 ubiquitin ligase of TPIT. Depending on the RING domain, TRIM65 ubiquitinated and degraded the TPIT protein at multiple Lys sites. In addition, TRIM65-mediated ubiquitination of TPIT inhibited POMC transcription and ACTH production to determine the fate of the novel subtype of PitNETs in vitro and in vivo. CONCLUSION: Our studies provided a novel classification of PitNETs and revealed that the TRIM65-TPIT complex controlled the fate of the novel subtype of PitNETs, which provides a potential therapy target for Cushing's disease.
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Proteínas de Homeodomínio , Tumores Neuroendócrinos , Neoplasias Hipofisárias , Proteínas com Domínio T , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Hormônio Adrenocorticotrópico/genética , Hormônio Adrenocorticotrópico/metabolismo , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Tumores Neuroendócrinos/patologia , Hipersecreção Hipofisária de ACTH , Neoplasias Hipofisárias/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Dioscin, an extract from traditional Chinese herbal plants, displays various biological and pharmacological effects on tumors, including inhibition of cell proliferation and induction of DNA damage. However, the effects of dioscin on oral squamous cell carcinoma (OSCC) cells are not completely understood. The present study aimed to evaluate the impact of dioscin on OSCC cell proliferation. Cell Counting Kit8 and 5ethynyl2'deoxyuridine incorporation assays were performed to assess cell proliferation. Flow cytometry was conducted to detect alterations in the cell cycle and cell apoptosis. Western blotting and coimmunoprecipitation were performed to determine protein expression levels. In SCC15 cells, dioscin treatment significantly induced cell cycle arrest, increased apoptosis and inhibited proliferation compared with the control group. Mechanistically, the tumor suppressor protein Ras association domaincontaining protein 1A (RASSF1A) was activated and oncoprotein yesassociated protein (YAP) was phosphorylated by dioscin. Furthermore, YAP overexpression and knockdown reduced and enhanced the inhibitory effects of dioscin on SCC15 cells, respectively. In summary, the results demonstrated that, compared with the control group, dioscin upregulated RASSF1A expression in OSCC cells, which resulted in YAP phosphorylation, thus weakening its transcriptional coactivation function, enhancing cell cycle arrest and apoptosis, and inhibiting cell proliferation. The present study indicated that dioscin may serve as a therapeutic agent for OSCC.
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Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células/efeitos dos fármacos , Diosgenina/análogos & derivados , Neoplasias Bucais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Diosgenina/farmacologia , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinase 3 , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
Metformin is an antidiabetic drug that has been reported to have an inhibitory effect on different types of cancers, including oral squamous cell carcinoma (OSCC). However, few studies have explored the role of Yesassociated protein (YAP), a vital factor contributing to OSCC biology, in metformininduced anticancer activity in OSCC cells. Thus, the purpose of the present study was to investigate the molecular relationship between metformin and YAP in OSCC cells. CAL27 and SCC25 cell lines were treated with various concentrations of metformin for 24 h. Cell proliferation was detected by Cell Counting Kit8 (CCK8) and flow cytometric assays. Cell apoptosis was analyzed using flow cytometry. The intracellular protein levels of target genes were determined by western blotting. It was demonstrated that metformin affected the cell cycle and apoptosis of CAL27 and SCC25 cells. Alteration of YAP protein expression affected metforminmediated changes in the cell cycle and apoptosis of CAL27 and SCC25 cells. In addition, compared to the control treatment, metformin treatment decreased the protein levels of YAP, mTOR, pmTOR and cMyc. The overexpression of YAP alleviated the inhibitory effect of metformin on the protein expression of mTOR, pmTOR and cMyc. The combination of metformin and verteporfin markedly enhanced the effects of metformin on the protein expression of mTOR, pmTOR and cMyc. Therefore, the results of the present study suggest that metformin suppresses OSCC by inhibiting YAP protein expression and by suppressing the YAPmediated effects of metformin on the protein expression of mTOR and cMyc.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Metformina/farmacologia , Neoplasias Bucais/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Bucais/patologia , Fosforilação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Elevated lipogenesis is an important metabolic hallmark of rapidly proliferating tumor such as endometrial carcinoma (EC). The sterol regulatory element-binding protein 1 (SREBP1) is a master regulator of lipogenesis and involved in EC proliferation. BF175 is a novel chemical inhibitor of SREBP pathway, and has shown potent anti-lipogenic effects. However, the effect of BF175 on EC cells are yet to be determined. In the present study, we found that BF175 decreased cell viability, colony formation and migratory capacity, inducing autophagy and mitochondrial related apoptosis in EC cell line AN3CA. Z-VAD-FMK partially attenuated the effect of BF175 on AN3CA. In addition, BF175 significantly downregulated SREBPs and their downstream genes. The levels of free fatty acids and total cholesterol were also inhibited. Microarray analysis suggested BF175 treatment obviously affected lipid metabolic pathways in EC. Taken together, we validated BF175 exhibited anti-tumor activity by targeting SREBP-dependent lipogenesis and inducing apoptosis which mitochondrial pathway involved in, suggesting that it's potential as a novel therapeutic reagent for EC.
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Compostos de Boro/farmacologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Redes e Vias Metabólicas , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ácidos Graxos/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ensaio Tumoral de Célula-TroncoRESUMO
Pituitary adenomas (PAs) are common, benign intracranial tumors that are usually effectively controlled with surgery, pharmacotherapy or radiotherapy. Some PAs against which conventional treatment is ineffective are great clinical challenges at present. Autophagy is a widespread physiological process in cells. Through autophagy, cells can degrade damaged or redundant proteins and organelles and achieve the recycling of intracellular substances to maintain the homeostasis of the intracellular environment. An increasing number of studies have demonstrated the importance of autophagy in tumor therapy. Both radiotherapy and chemotherapy can induce autophagy, which plays different roles in the course of therapy. In recent years, there has been growing interest in the role of autophagy during the treatment of PAs. This chapter reviews the recent progress of research on autophagy in PA and the autophagic mechanisms in the treatment of PA.
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Adenoma , Autofagia , Neoplasias Hipofisárias , Humanos , Hipófise/patologiaRESUMO
OBJECTIVES: Long non-coding RNA (lncRNA) MATN1-AS1 is a newfound lncRNA that has been rarely explored in cancers. Herein, we would like to investigate its role in glioma. MATERIALS AND METHODS: qRT-PCR was conducted to examine gene expression in glioma. Then, MTT assay, colony formation assay and flow cytometry analysis were applied to evaluate the function of MATN1-AS1 on glioma cells. Western blot was performed to measure the protein levels of genes. Besides, the luciferase reporter assay, RNA pull-down assay, RIP assay and Spearman's correlation analysis were also performed as needed. RESULTS: Firstly, a data from TCGA showed that MATN1-AS1 might be largely implicated in glioma. Meanwhile, MATN1-AS1 upregulation confirmed in glioma predicted poor clinical outcomes. Functionally, MATN1-AS1 knockdown restrained cell proliferation but stimulated apoptosis in vitro and repressed tumour growth in vivo. Mechanistic investigations validated that MATN1-AS1 functioned as a ceRNA for miR-200b/c/429 to upregulate CHD1 which was also verified to exert a growth-promoting role in glioma cells here. Importantly, both CHD1 overexpression and miR-200b/c/429 inhibition could rescue the obstructive role of MATN1-AS1 silence in glioma cells. CONCLUSIONS: MATN1-AS1 promotes glioma progression through regulating miR-200b/c/429-CHD1 axis, suggesting MATN1-AS1 as a probable target for glioma treatment.
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DNA Helicases/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Idoso , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Feminino , Glioma/genética , Glioma/patologia , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genéticaRESUMO
OBJECTIVE: This study aims to offer insights about the biological influence of TAZ, which is a transcriptional coactivator containing a PDZ-binding motif, upon the apoptosis, proliferation, and osteogenic differentiation of human periodontal ligament stem cells (h-PDLSCs). METHODS: We used the green fluorescence protein lentivirus infection system to knockdown or overexpress TAZ in h-PDLSCs. 5-ethynyl-2'-deoxyuridine (EdU) staining detected the proliferative activity, and h-PDLSC apoptosis was analyzed by Annexin V-APC staining. TAZ knockdown or overexpression was performed to determine the osteogenic differentiation function of TAZ during the osteogenic induction of h-PDLSCs. The molecular mechanism of TAZ in the promotion of h-PDLSC osteogenesis was also explored. The chemical inhibitor of SMAD2/3 SIS3 HCL was used to identify the effects in vitro osteogenic differentiation and bone formation in h-PDLSCs overexpressing TAZ. RESULTS: TAZ overexpression resulted in enhanced cell rapid multiplication, which increased the expression of messenger RNA in stemness-related genes. By comparison, TAZ knockdown reduced proliferative activity and increased the apoptosis of h-PDLSCs. After the 7-day osteogenic induction period, alkaline phosphatase activity in the TAZ-overexpression group was significantly increased, and mineralized nodules increased significantly after osteogenic induction for 21 days. Similarly, osteoblast differentiation of h-PDLSCs was impaired after TAZ knockdown. However, the osteogenic potential of the group exposed to the p-SMAD3 inhibitor was restored to its original level. CONCLUSION: Hippo/TAZ plays a positive role inside the proliferation, stemness maintenance, and osteogenic specialization of h-PDLSCs, and the specific downstream factor of osteogenic differentiation is SMAD3.
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Diferenciação Celular , Proliferação de Células , Osteogênese , Ligamento Periodontal/metabolismo , Células-Tronco/metabolismo , Transativadores/biossíntese , Humanos , Ligamento Periodontal/citologia , Células-Tronco/citologia , Transativadores/genética , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
OBJECTIVE: To establish an immortalized human periodontal ligament stem cell line (hPDLSC) and investigate whether and how YAP mediates the establishment of the stem cell line. METHODS: Primary hPDLSCs were cultured and transfected with lentivirus containing the telomerase reverse transcriptase (TERT) gene. The expression of TERT was detected via the polymerase chain reaction (PCR) and real-time quantitative PCR (RT-PCR). Flow cytometry was employed to detect surface markers of hPDLSCs and TERT-hPDLSCs. The cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) methods were used to examine the proliferation ability of the cells. Flow cytometry and TUNEL staining were employed to examine the cell apoptosis rate. The ß-galactosidase staining assay was used to assess the rate of cell senescence. The osteogenic differentiation ability of the cells was detected via alkaline phosphatase (ALP) staining and Alizarin red staining assays. BALB/c mice were employed to determine the tumorigenicity of TERT-hPDLSCs. The expression levels of YAP and other proteins in the Hippo signaling pathway were detected by Western blotting. Verteporfin was used to inhibit the binding of YAP to the downstream target gene TEAD. RESULTS: TERT-hPDLSCs showed stable high expression of TERT, even at the thirtieth passage after transfection with lentivirus containing the TERT gene. Compared with primary hPDLSCs, TERT-hPDLSCs exhibited a stronger proliferation ability and lower cell apoptosis and senescence rates while maintaining the same osteogenetic differentiation ability as primary hPDLSCs. The transfection of hPDLSCs with lentivirus containing the TERT gene did not lead to tumorigenesis in nude mice. The Hippo signaling pathway was inactivated in TERT-hPDLSCs compared to hPDLSCs. When treated with verteporfin, the proliferation of TERT-hPDLSCs decreased, while the apoptosis and senescence rates of these cells increased. However, TERT-hPDLSCs still showed a stronger proliferation ability and lower cell apoptosis and senescence rates than hPDLSCs treated with verteporfin at the same concentration. CONCLUSIONS: Overexpression of TERT in hPDLSCs resulted in the successful establishment of an immortalized periodontal ligament stem cell line. TERT may regulate the biological characteristics of hPDLSCs through the Hippo/YAP signaling pathway. hPDLSCs could be a feasible resource for stem cell research and a promising resource for stem cell therapy.
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Objectives: To provide insight into the biological effects of activated Yes-associated protein (YAP) on the proliferation, apoptosis, and senescence of human periodontal ligament stem cells (h-PDLSCs). Methods: h-PDLSCs were isolated by the limiting dilution method, and their surface markers were quantified by flow cytometry. Enhanced green fluorescence protein (EGFP)-labeled lentiviral vector was used to activate YAP in h-PDLSCs, then qRT-PCR and Western blotting were used to evaluate the expression level of YAP. Immunofluorescence was used to detect the location of YAP in h-PDLSCs. The proliferation activity was detected by cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU), and the cell cycle was determined by flow cytometry. Apoptosis was analyzed by Annexin V-APC staining. Cell senescence was detected by ß-galactosidase staining. Proteins in ERK, Bcl-2, and p53 signaling pathways were detected by Western blotting. Results: h-PDLSCs were isolated successfully and were positive for human mesenchymal stem cell surface markers. After YAP was activated by lentiviral vector, the mRNA and protein of YAP were highly expressed, and more YAP translocated into the nucleus. When YAP was overexpressed in h-PDLSCs, proliferation activity was improved; early and late apoptosis rates decreased (P<0.05); the proportion of cells in G2/M phases increased (P<0.05), while that in G0/G1 phase decreased (P<0.05); cellular senescence was delayed (P<0.01); the expression of P-MEK, P-ERK, P-P90RSK and P-Msk increased, while the expression of Bcl-2 family members (Bak, Bid and Bik) decreased. Conclusions: Activated YAP promotes proliferation, inhibits apoptosis, and delays senescence of h-PDLSCs. The Hippo-YAP signaling pathway can influence ERK and Bcl-2 signaling pathways.
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Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose , Senescência Celular , Ligamento Periodontal/fisiologia , Fosfoproteínas/fisiologia , Células-Tronco/fisiologia , Adolescente , Diferenciação Celular , Proliferação de Células , Criança , Humanos , Osteogênese , Ligamento Periodontal/citologia , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Transcriptional co-activator Yes-associated protein (YAP) is a key oncogene in mammalian cells. The present understanding of YAP in oral squamous cells carcinoma (OSCC) remains unclear. The purpose of this study is to investigate the effects of YAP on proliferation and apoptosis in OSCC and the molecular mechanism. The results showed the expression level of YAP was higher in OSCC tissues than that in adjacent normal tissues. Knockdown of YAP in CAL27 cell lines prohibited cell proliferation while augmented apoptosis. Conversely, overexpression of YAP protected cells from apoptosis and promoted cell proliferation. Moreover, C-MYC and BCL-2 mRNA and protein levels were altered due to the differential expression of YAP. Subsequent Verteporfin treatment in CAL27 cells revealed that the transcription and translation of BCL-2 and C-MYC both decreased. In a tumor xenograft model, knockdown of YAP suppressed tumor growth of CAL27 in vivo, while YAP overexpression promoted the tumor growth. These results suggest that YAP is a crucial regulator that exerts pro-proliferation and anti-apoptosis effects in OSCC through actions affecting the cell cycle and intrinsic apoptotic signaling. Thus YAP could potentially serve as a valuable molecular biomarker or therapeutic target in the treatment of OSCC.
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Objectives: The present study established a non-contact coculture system in vitro, aiming to investigate the crosstalk between human dental pulp stem cells (hDPSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation activity and osteogenic genes expression through paracrine. Materials and methods: The stemness of hDPSCs and hUCMSCs were identified by flow cytometric analysis and multipotential differentiation assays. With the help of transwell inserts, the non-contact coculture system in vitro was established between hDPSCs and hUCMSCs. EdU labeling analysis and Western Blot were used to detect the proliferation activity. The mRNA and protein levels of osteogenic genes were evaluated by RT-PCR and Western Blot. The expression of elements in Akt/mTOR signaling pathway were detected by Western Blot. Results: Both hDPSCs and hUCMSCs were positive to MSCs specific surface markers and had multi-differentiation potential. The proportion of EdU-positive cells increased and the expression of CDK6 and CYCLIN A were up-regulated in cocultured hDPSCs. Both prior coculture and persistent coculture improved mRNA and protein levels of osteogenic genes in hDPSCs. While in cocultured hUCMSCs, no statistical differences were observed on proliferation and osteogenesis. The phosphorylation of Akt and mTOR was up-regulated in cocultured hDPSCs. Conclusions: The crosstalk between hDPSCs and hUCMSCs in coculture system increased the proliferation activity and enhanced osteogenic genes expression in hDPSCs. Akt/mTOR signaling pathway might take part in the enhancing effects in both cell proliferation and gene expression.
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Técnicas de Cocultura/métodos , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacosRESUMO
Objective: The purpose of this study was to provide an insight into the biological effects of knockdown Yes-associated protein (YAP) on the proliferation and apoptosis of human periodontal ligament stem cells (h-PDLSCs). Methods: Immunofluorescence and Western blot were used to evaluate Hippo-YAP signaling expression level. Enhanced green fluorescence protein lentiviral vector was constructed to down-regulate YAP in h-PDLSCs. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect the interfering efficiency of YAP expression. The proliferation activity was detected by EdU staining. Analysis of apoptosis in h-PDLSCs was done through Annexin V-APC staining, while cell cycle analysis was detected by flow cytometry. Cellular senescence was analyzed by ß-galactosidase activity detection. The expression of elements in signaling pathways related with proliferation and apoptosis was detected by Western blot. Results: YAP was located in nucleus and cytoplasm. After the lentivirus transfection, the expression of YAP mRNA and protein was significantly reduced (P<0.001). When YAP was knocked down, the proliferation activity of h-PDLSCs was inhibited; the early & late apoptosis rates increased; the proportion of cells in G1 phases increased (P<0.05), while that in G2 and S phase decreased (P<0.05); cellular senescence was accelerated (P<0.01); ERK and its target proteins P-P90RSK and P-MEK were reduced while Bcl-2 family members increased. Conclusion: Knockdown of YAP inhibits the proliferation activity and induces apoptosis of h-PDLSCs with the involvement of Hippo pathway and has a crosstalk between Erk and Bcl-2 signaling pathways.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proliferação de Células , Sistema de Sinalização das MAP Quinases , Fosfoproteínas/metabolismo , Células-Tronco/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Via de Sinalização Hippo , Humanos , Ligamento Periodontal/citologia , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro , RNA Interferente Pequeno , Fatores de Transcrição , Transfecção , Proteínas de Sinalização YAPRESUMO
Collagen triple helix repeat containing 1 (CTHRC1) is associated with bone metabolism. Alveolar bone has an ability to rapidly remodel itself to adapt its biomechanical environment and function. However, whether CTHRC1 is expressed in alveolar bone tissue and the role of CTHRC1 in alveolar bone remodeling remain unclear. We used orthodontic tooth movement (OTM) rat model to study the effects of CHTRC1 in alveolar bone remodeling in vivo. We found that CTHRC1 was expressed in normal physiological condition of osteocytes, bone matrix, and periodontal ligament cells in rat. During the OTM, the expression of CTHRC1, Runx2 and TAZ were increased. We further studied the effects of CTHRC1 on osteogenic differentiation of human periodontal ligament stem cells in vitro. CTHRC1 can positively regulate the expression of TAZ and osteogenic differentiation markers like Col1, ALP, Runx2 and OCN. Overexpression of CHTRC1 increased osteogenic differentiation of PDLSCs, which could be abolished by TAZ siRNA. Our results suggest that CTHRC1 plays an important role in alveolar bone remodeling and osteogenic differentiation of PDLSCs.
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Diferenciação Celular , Glicoproteínas/fisiologia , Osteogênese , Ligamento Periodontal/citologia , Células-Tronco/citologia , Fatores de Transcrição/fisiologia , Aciltransferases , Processo Alveolar , Animais , Remodelação Óssea , Regulação da Expressão Gênica , Humanos , Ratos , Técnicas de Movimentação DentáriaRESUMO
Surgery, chemotherapy, and radiotherapy are the three top cancer treatment modalities. Paclitaxel (PTX) is one of the most widely used chemotherapy drugs. However, its clinical applications have been significantly limited due to: (i) serious hemolysis effect of currently available commercial paclitaxel formulations and (ii) its water insolubility. An easy way to deliver paclitaxel by a new nanocarrier system using pluronic copolymers of P123/F68 and Sorbitan monopalmitate (Span 40) was reported in our previous research article. The characterization of the formulation and analysis of drug release and cellular uptake were also presented. In this article, we reported discoveries of our follow-up in vivo antitumor and in vitro hemolytic study discoveries. The experimental results showed that the nanoformulated PTX achieved much better tumor suppression performance while reducing hemolysis side effects. This newly formulated drug can significantly improve patient outcomes in cancer chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos/química , Hexoses/química , Neoplasias/tratamento farmacológico , Paclitaxel/administração & dosagem , Poloxâmero/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Feminino , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Camundongos Nus , Micelas , Neoplasias/patologia , Paclitaxel/química , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Solubilidade , Água/químicaRESUMO
Cigarette smoking is one of highly risk factors of cervical cancer. Recently nicotine has been reported to increase proliferation and invasion in some smoking related cancers, like non-small cell lung cancer and esophageal squamous cell cancer. However, the effects and mechanisms of nicotine stimulation on cervical cancer cells are not clear. Here, we investigated the effects and mechanisms of nicotine stimulation on HeLa cells in vitro. In our study, we found that nicotine could accelerate HeLa cells migration and invasion, activate PI3K/Akt and NF-κB pathways and increase the expression of Vimentin in vitro. Moreover, we demonstrated that the specific PI3K inhibitor LY294002 could reverse nicotine-induced cell migration and invasion, NF-κB activation and up-regulation of Vimentin. Inhibition of NF-κB by Pyrrolidine dithiocarbamate (PDTC) also antagonized nicotine-induced cell migration, invasion and up-regulation of Vimentin. Simply put, these findings suggest that nicotine promotes cervical carcinoma cell line HeLa migration and invasion by activating PI3k/Akt/NF-κB pathway in vitro.
Assuntos
Movimento Celular/efeitos dos fármacos , Nicotina/toxicidade , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/patologia , Feminino , Células HeLa , Humanos , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Invasividade Neoplásica/patologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
This current study tested expression and potential biological functions of Gab3 in human glioma. Gab3 mRNA and protein expression was significantly elevated in human glioma tissues and glioma cells. Its level was however low in normal brain tissues and primary human astrocytes. In both established (U251MG cell line) and primary human glioma cells, Gab3 knockdown by shRNA/siRNA significantly inhibited Akt activation and cell proliferation. Reversely, forced Gab3 overexpression in U251MG cells promoted Akt activation and cell proliferation. In vivo, the growth of U251MG tumors in nude mice was inhibited following expressing Gab3 shRNA. Akt activation in cancer tissues was also suppressed by Gab3 shRNA. Together, we conclude that Gab3 overexpression in human glioma mediates Akt activation and cancer cell proliferation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Glioma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias Encefálicas/patologia , Ativação Enzimática , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
Glioblastoma multiforme and anaplastic astrocytoma are challenges to clinical biologists at present. The patients with glioblastoma have median survival of less than 12 months, despite advances in radiotherapeutical, chemotherapeutical and conventional surgical modalities. Retinoic acids are known to effect in vitro proliferation, differentiation, and apoptosis in colon, prostate, lung, and leukemia cancers. Retinoids are known to have anti-proliferation, anti-migration, and anti-invasive activity against human malignant gliomas, suggesting that retinoids are suitable anticancer agents to inhibit progression of tumors. Recurrent malignant cerebral gliomas have been treated with ATRA and 13-cis RA. However, the side effects associated with the use of high doses of retinoic acid demand for some more potent derivative free from such effects. The present clinical trials are undertaken to investigate the clinical safety and possible efficacy of administering retinoic acid naphthalene triazole (RANT) to patients with recurrent malignant gliomas. The toxicities observed in the patients during RANT treatment were mild. These preliminary results suggest that RANT is more potent compared to RA against recurrent malignant gliomas.