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1.
PLoS One ; 15(9): e0239456, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32976517

RESUMO

The use of ultrasound-stimulated microbubble therapy has successfully been used to target tumor vasculature and enhance the effects of radiation therapy in tumor xenografts in mice. Here, we further investigate this treatment using larger, more clinically relevant tumor models. New Zealand white rabbits bearing prostate tumor (PC3) xenografts received a single treatment of either ultrasound-stimulated microbubbles (USMB), ionizing radiation (XRT; 8Gy), or a combination of both treatments (USMB+XRT). Treatment outcome was evaluated 24 hours after treatment using histopathology, immunolabeling, 3D Doppler ultrasound and photoacoustic imaging. A second cohort of rabbits received multiple treatments over a period of three weeks, where USMB treatments were delivered twice weekly with daily XRT treatments to deliver a fractionated 2Gy dose five days per week. A significant decrease in vascular function, observed through immunolabeling of vascular endothelial cells, was observed in tumors receiving the combined treatment (USMB+XRT) compared to control and single treatment groups. This was associated with an increase in cell death as observed through in situ end labeling (ISEL), a decrease in vascular index measured by Power Doppler imaging, and a decrease in oxygen saturation. In rabbits undergoing the long-term fractionated combined treatment, a significant growth delay was observed after 1 week and a significant reduction in tumor size was observed after 3 weeks with combined therapy. Results demonstrated an enhancement of radiation effect and superior anti-tumor effect of the combination of USMB+XRT compared to the single treatments alone. Tumor growth was maximally inhibited with fractionated radiotherapy combined with the ultrasound-stimulated microbubble-based therapy.


Assuntos
Microbolhas/uso terapêutico , Neoplasias da Próstata/radioterapia , Terapia por Ultrassom/métodos , Animais , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Terapia Combinada/métodos , Células Endoteliais/efeitos da radiação , Humanos , Masculino , Camundongos , Células PC-3 , Coelhos , Ondas Ultrassônicas
2.
Tumour Biol ; 42(9): 1010428320957506, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32914709

RESUMO

The development of the multidrug resistance phenotype is one of the major challenges faced in the treatment of cancer. The multidrug resistance phenotype is characterized by cross-resistance to drugs with different chemical structures and mechanisms of action. In this work, we hypothesized that the acquisition of resistance in cancer is accompanied by activation of the epithelial-to-mesenchymal transition process, where the tumor cell acquires a more mobile and invasive phenotype; a fundamental step in tumor progression and in promoting the invasion of other organs and tissues. In addition, it is known that atypical glycosylations are characteristic of tumor cells, being used as biomarkers. We believe that the acquisition of the multidrug resistance phenotype and the activation of epithelial-to-mesenchymal transition provoke alterations in the cell glycophenotype, which can be used as glycomarkers for chemoresistance and epithelial-to-mesenchymal transition processes. Herein, we induced the multidrug resistance phenotype in the PC-3 human prostate adenocarcinoma line through the continuous treatment with the drug paclitaxel. Our results showed that the induced cell multidrug resistance phenotype (1) acquired a mixed profile between epithelial and mesenchymal phenotypes and (2) modified the glycophenotype, showing an increase in the level of sialylation and in the number of branched glycans. Both mechanisms are described as indicators of poor prognosis.


Assuntos
Adenocarcinoma/patologia , Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Paclitaxel/farmacologia , Adenocarcinoma/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Glicosilação , Humanos , Células PC-3 , Fenótipo
3.
J Hematol Oncol ; 13(1): 120, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887634

RESUMO

BACKGROUND: Critically ill patients diagnosed with COVID-19 may develop a pro-thrombotic state that places them at a dramatically increased lethal risk. Although platelet activation is critical for thrombosis and is responsible for the thrombotic events and cardiovascular complications, the role of platelets in the pathogenesis of COVID-19 remains unclear. METHODS: Using platelets from healthy volunteers, non-COVID-19 and COVID-19 patients, as well as wild-type and hACE2 transgenic mice, we evaluated the changes in platelet and coagulation parameters in COVID-19 patients. We investigated ACE2 expression and direct effect of SARS-CoV-2 virus on platelets by RT-PCR, flow cytometry, Western blot, immunofluorescence, and platelet functional studies in vitro, FeCl3-induced thrombus formation in vivo, and thrombus formation under flow conditions ex vivo. RESULTS: We demonstrated that COVID-19 patients present with increased mean platelet volume (MPV) and platelet hyperactivity, which correlated with a decrease in overall platelet count. Detectable SARS-CoV-2 RNA in the blood stream was associated with platelet hyperactivity in critically ill patients. Platelets expressed ACE2, a host cell receptor for SARS-CoV-2, and TMPRSS2, a serine protease for Spike protein priming. SARS-CoV-2 and its Spike protein directly enhanced platelet activation such as platelet aggregation, PAC-1 binding, CD62P expression, α granule secretion, dense granule release, platelet spreading, and clot retraction in vitro, and thereby Spike protein enhanced thrombosis formation in wild-type mice transfused with hACE2 transgenic platelets, but this was not observed in animals transfused with wild-type platelets in vivo. Further, we provided evidence suggesting that the MAPK pathway, downstream of ACE2, mediates the potentiating role of SARS-CoV-2 on platelet activation, and that platelet ACE2 expression decreases following SARS-COV-2 stimulation. SARS-CoV-2 and its Spike protein directly stimulated platelets to facilitate the release of coagulation factors, the secretion of inflammatory factors, and the formation of leukocyte-platelet aggregates. Recombinant human ACE2 protein and anti-Spike monoclonal antibody could inhibit SARS-CoV-2 Spike protein-induced platelet activation. CONCLUSIONS: Our findings uncovered a novel function of SARS-CoV-2 on platelet activation via binding of Spike to ACE2. SARS-CoV-2-induced platelet activation may participate in thrombus formation and inflammatory responses in COVID-19 patients.


Assuntos
Betacoronavirus/metabolismo , Plaquetas/metabolismo , Infecções por Coronavirus/metabolismo , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Trombose/metabolismo , Adulto , Idoso , Animais , Betacoronavirus/genética , Células CACO-2 , Infecções por Coronavirus/virologia , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Células PC-3 , Pandemias , Peptidil Dipeptidase A/genética , Agregação Plaquetária/imunologia , Contagem de Plaquetas , Pneumonia Viral/virologia , RNA Viral/sangue , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Trombose/virologia
4.
Life Sci ; 258: 118232, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781066

RESUMO

AIMS: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. MAIN METHODS: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. KEY FINDINGS: EGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 µM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. SIGNIFICANCE: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.


Assuntos
Antioxidantes/farmacologia , Cálcio/metabolismo , Catequina/análogos & derivados , Líquido Intracelular/metabolismo , Neoplasias da Próstata/metabolismo , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Líquido Intracelular/efeitos dos fármacos , Masculino , Células PC-3
5.
Gene ; 762: 145034, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777521

RESUMO

Carbonic Anhydrase III (CAIII) belongs to a member of the alpha Carbonic Anhydrase (CA) family. Although some CA members are strongly up-regulated by HIF1-α, it is not known about the transcriptional regulation of CAIII in prostate cancer cells, PCa. Therefore, we aimed to identify regulatory regions important for the regulation of CAIII gene under hypoxic conditions in human prostate cancer cells (PC3). The present study, for the first time, demonstrated that the chemically mimicked hypoxic condition led to the induced CAIII mRNA and protein expression in prostate cancer cells. Transcriptional regulation of CAIII was investigated by transient transfection assay that indicates that the most active promoter activity was in the region of P2 -699/+86. Hypoxic condition also upregulates the basal activity of for P1;-941/+86 and P2;-699/+86 constructs containing putative Hypoxia Response Element (HRE) region located in -268/-252. EMSA analysis of HRE located in -268/-252 bases, showed one DNA-protein binding complexes. Competition assays indicated this complex is resulted from HIF1α interactions. In addition, site-directed mutagenesis of potential HIF1α binding sites diminished a DNA-protein complex. These findings suggest that CAIII is a hypoxia-regulated gene and valuable for targeting of prostate cancer tumors in hypoxic condition.


Assuntos
Anidrase Carbônica III/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Próstata/metabolismo , Anidrase Carbônica III/metabolismo , Hipóxia Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Células PC-3 , Regiões Promotoras Genéticas , Regulação para Cima
6.
Nat Cell Biol ; 22(9): 1130-1142, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32839549

RESUMO

Epigenetic plasticity is a pivotal factor that drives metastasis. Here, we show that the promoter of the gene that encodes the ubiquitin ligase subunit FBXL7 is hypermethylated in advanced prostate and pancreatic cancers, correlating with decreased FBXL7 mRNA and protein levels. Low FBXL7 mRNA levels are predictive of poor survival in patients with pancreatic and prostatic cancers. FBXL7 mediates the ubiquitylation and proteasomal degradation of active c-SRC after its phosphorylation at Ser 104. The DNA-demethylating agent decitabine recovers FBXL7 expression and limits epithelial-to-mesenchymal transition and cell invasion in a c-SRC-dependent manner. In vivo, FBXL7-depleted cancer cells form tumours with a high metastatic burden. Silencing of c-SRC or treatment with the c-SRC inhibitor dasatinib together with FBXL7 depletion prevents metastases. Furthermore, decitabine reduces metastases derived from prostate and pancreatic cancer cells in a FBXL7-dependent manner. Collectively, this research implicates FBXL7 as a metastasis-suppressor gene and suggests therapeutic strategies to counteract metastatic dissemination of pancreatic and prostatic cancer cells.


Assuntos
Epigênese Genética/genética , Transição Epitelial-Mesenquimal/genética , Proteínas F-Box/genética , Inativação Gênica/fisiologia , Metástase Neoplásica/genética , Subunidades Proteicas/genética , Quinases da Família src/genética , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células PC-3 , Transdução de Sinais/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
7.
Gene ; 763: 145067, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32827681

RESUMO

BACKGROUND: rs2274911 (Pro91Ser, G > A) is a missense mutation located on the second exon of the GPRC6A gene. Increasing evidence revealed a significant association between the A allele of rs2274911 and male diseases, such as oligospermia, cryptorchidism, and prostate tumor. However, the function of rs2274911 in healthy males is unclear. SUBJECTS AND METHODS: A total of 1742 healthy men were selected from the Fangchenggang Area Male Health and Examination Survey (FAMHES). The association between rs2274911 and phenotype was evaluated. The cell characteristics of rs2274911 mutation (mu), wild-type GPRC6A (WT), and RFP control in human embryonic kidney (293T) and human prostate cancer (PC3) cells were analyzed. RNA sequencing was performed on PC3 cells. RESULTS: E2 and PSA serum levels increased with the accumulation of the A allele (E2: G vs. A, -0.029 [-0.050, -0.008], P < 0.01, P trend = 0.027; PSA: G vs. A, -0.040 [-0.079, 0.000], P < 0.05, P trend = 0.048). rs2274911 enhanced the proliferation and invasion ability of PC3 or 293T cells and activated the ERK pathway. The genes were identified as rs2274911 mu-affected genes through RNA sequential analysis of rs2274911 mu, GPRC6A WT, and RFP control of PC3 cells. Most of these genes were related to cancer development processes, cAMP, and the ERK cell signaling pathway. CONCLUSION: This project represents that rs2274911 is associated with E2 and PSA serum levels in Southern Chinese men. Rs2274991 mutation promotes 293T and PC3 cell proliferation in vitro. These results suggest that rs2274911 is a functional variant of GPRC6A.


Assuntos
Antígeno 12E7/sangue , Polimorfismo de Nucleotídeo Único , Antígeno Prostático Específico/sangue , Receptores Acoplados a Proteínas-G/genética , Adulto , Proliferação de Células , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Células PC-3 , Receptores Acoplados a Proteínas-G/metabolismo
8.
Prostate ; 80(12): 1024-1037, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32628792

RESUMO

BACKGROUND: Dysregulation of microRNAs has performed vital gene regulatory functions in the genesis, progression, and prognosis of multiple malignant tumors. This study aimed to elucidate the regulatory mechanism of miR-196a in prostate cancer (PCa) and explore its clinical significance. METHODS: Quantitative real-time polymerase chain reaction was implemented to examine miR-196a and p27kip1 messenger RNA expression in PCa. Cell proliferation was evaluated via Cell Counting Kit-8, colony formation, and nude mouse tumorigenicity assays. Luciferase reporter assay was applied to identify target genes. p27kip1 protein expression in PCa was investigated using Western blot analysis and immunohistochemistry. RESULTS: There was a dramatic upregulation of miR-196a in PCa. Upregulated miR-196a was related to worse Gleason score (GS), later pathological stage, and poor biochemical recurrence (BCR)-free survival. In vivo and in vitro experiments exhibited that miR-196a promoted PCa proliferation and expedited G1/S-phase progression through the downregulation of p27kip1 protein. Additionally, p27kip1 protein was distinctly downregulated in PCa. Low p27kip1 protein expression had a strong correlation with increased GS and was an independent predictor of BCR after radical prostatectomy (RP). CONCLUSIONS: Excessive expression of miR-196a and subsequent downregulation of p27kip1 protein play essential roles in promoting PCa proliferation and leading to BCR after RP. miR-196a and its target p27kip1 may become novel molecular biomarkers and therapeutic targets for PCa.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação para Baixo , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Células PC-3 , Prostatectomia/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia
9.
Prostate ; 80(13): 1087-1096, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32609927

RESUMO

BACKGROUND: Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. METHODS: Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. RESULTS: ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. CONCLUSIONS: This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.


Assuntos
Tratamento por Ondas de Choque Extracorpóreas/métodos , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Células Estromais/patologia , Actinas/genética , Actinas/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Progressão da Doença , Humanos , Masculino , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/metabolismo
10.
Prostate ; 80(13): 1134-1144, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32628304

RESUMO

BACKGROUND: Although androgen deprivation therapy (ADT) is the initial treatment strategy for prostate cancer (PCa), recurrent castration-resistant prostate cancer (CRPC) eventually ensues. In this study, cancer-derived immunoglobulin G (CIgG) is found to be induced after ADT, identifying CIgG as a potential CRPC driver gene. METHODS: The expression of CIgG and its clinical significance in PCa tissue was analyzed by The Cancer Genome Atlas database and immunohistochemistry. Subsequently, the sequence features of prostate cell line VHDJH rearrangements were analyzed. We also assessed the effect of CIgG on the migratory, invasive and proliferative abilities of PCa cells in vitro and vivo. Suspended microsphere, colony formation and drug-resistant assays were performed using PC3 cells with high CIgG expression (CIgGhigh ) and low CIgG expression (CIgG-/low ), and A nonobese diabetic/severe combined immunodeficiency mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. The SOX2-CIgG signaling pathway was validated by immunohistochemistry, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, Western blot, luciferase, and chromatin immunoprecipitation assays and bioinformatics analyses. Finally, we investigated the effect of RP215 inhibition on the progression of PCa in vivo using a Babl/c nude mouse xenograft model. RESULTS: CIgG is frequently expressed in PCa and associated with clinicopathological characteristics, moreover, CIgG transcripts with unique patterns of VHDJH rearrangements are found in PCa cells. Functional analyses identified that CIgG was induced by ADT and upregulated by SOX2 (SRY (sex determining region Y)-box 2) in PCa, promoting the development of PCa. In addition, our findings underscore a novel role of CIgG signaling in the maintenance of stemness and the progression of cancer through mitogen activated protein kinase/extracellular-signal-regulated kinase and AKT in PCa. In vivo experiments further demonstrated that depleting CIgG significantly suppressed the growth of PCa cell xenografts. Furthermore, a CIgG monoclonal antibody named RP215 exhibits tumor inhibitory effect as well. CONCLUSION: Our data suggests that CIgG could be a driver of PCa development, and that targeting the SOX2-CIgG axis may therefore inhibit PCa development after ADT.


Assuntos
Imunoglobulina G/imunologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Fatores de Transcrição SOXB1/imunologia , Animais , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/imunologia , Análise Serial de Tecidos
11.
J Biol Regul Homeost Agents ; 34(3): 885-892, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32691575

RESUMO

We aimed to elucidate the inhibitory effect of Baicalein on proliferative ability in Prostate cancer (PCa) through downregulating Ezrin. Relative level of Ezrin in PCa tissues and adjacent ones was detected. After PC3 cells were induced with 20 or 40 µM Baicalein, changes in viability, cell cycle progression and apoptosis were assessed. Relative levels of CyclinD1, CDK4, P53 and P21 were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Regulatory effects of Ezrin and Baicalein treatment on PC3 cells were evaluated. Finally, in vivo effects of Ezrin and Baicalein treatment on nude mice bearing PCa were detected. Ezrin was upregulated in PCa tissues relative to adjacent normal ones. Baicalein treatment decreased viability, arrested cell cycle and stimulated apoptosis in PC3 cells. Meanwhile, Baicalein treatment downregulated CyclinD1 and CDK4, while upregulating P53 and P21. Moreover, Ezrin was downregulated in Baicalein-treated PC3 cells. Knockdown of Ezrin synergistically stimulated the effects of Baicalein on cellular phenotypes of PC3 cells. In nude mice bearing PCa, Baicalein treatment decreased tumor volume and tumor weight, which were much more pronounced in those with in vivo knockdown of Ezrin. Baicalein treatment suppresses proliferative ability, arrests cell cycle and stimulates apoptosis in PCa cells through downregulating Ezrin.


Assuntos
Apoptose , Neoplasias da Próstata , Animais , Linhagem Celular Tumoral , Proliferação de Células , Proteínas do Citoesqueleto , Flavanonas , Humanos , Masculino , Camundongos , Camundongos Nus , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética
12.
Nucleic Acids Res ; 48(19): 11130-11145, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32525981

RESUMO

Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein-RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.


Assuntos
Antígenos de Superfície/química , Aptâmeros de Nucleotídeos/química , Glutamato Carboxipeptidase II/química , Biomarcadores Tumorais/química , Células HEK293 , Humanos , Ligantes , Masculino , Estrutura Molecular , Células PC-3 , Neoplasias da Próstata/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
13.
Prostate ; 80(11): 885-894, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32483877

RESUMO

BACKGROUND: Eradication of persistent androgen receptor (AR) activity in castration-resistant prostate cancer may be a promising strategy to overcome castration resistance. We aimed to identify novel compounds that inhibit AR activity and could be potential therapeutic agents for prostate cancer. METHODS: A high-throughput screening system involving cell lines stably expressing AR protein and AR-responsive luciferase was employed for the 1260 compound library. Molecular and antitumor effects on candidate pathways that interacted with AR signaling were examined in prostate cancer cells expressing AR. RESULTS: The high-throughput screening identified various potential compounds that interfered with AR signaling through known and novel pathways. Among them, a 5-hydroxytryptamine 5A (5-HT5A) receptor antagonist suppressed AR activity through protein kinase A signaling, which was confirmed by 5-HT5A receptor knockdown. Consistently, 5-HT5A receptor inhibitors showed cytotoxic effects toward prostate cancer cells. CONCLUSIONS: Taken together, this study identifies 5-HT5A receptor as a promising therapeutic target for prostate cancer via its interaction with AR signaling.


Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo
14.
Prostate ; 80(12): 962-976, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32511787

RESUMO

OBJECTIVE: The broad goal of the research described in this study was to investigate the contributions of selenium-binding protein 1 (SBP1) loss in prostate cancer development and outcome. METHODS: SBP1 levels were altered in prostate cancer cell lines and the consequences on oxygen consumption, expression of proteins associated with energy metabolism, and cellular transformation and migration were investigated. The effects of exposing cells to the SBP1 reaction products, H2 O2 and H2 S were also assessed. In silico analyses identified potential HNF4α binding sites within the SBP1 promoter region and this was investigated using an inhibitor specific for that transcription factor. RESULTS: Using in silico analyses, it was determined that the promoter region of SBP1 contains putative binding sites for the HNF4α transcription factor. The potential for HNF4α to regulate SBP1 expression was supported by data indicating that HNF4α inhibition resulted in a dose-response increase in the levels of SBP1 messenger RNA and protein, identifying HNF4α as a novel negative regulator of SBP1 expression in prostate cancer cells. The consequences of altering the levels of SBP1 were investigated by ectopically expressing SBP1 in PC-3 prostate cancer cells, where SBP1 expression attenuated anchorage-independent cellular growth and migration in culture, both properties associated with transformation. SBP1 overexpression reduced oxygen consumption in these cells and increased the activation of AMP-activated protein kinase (AMPK), a major regulator of energy homeostasis. In addition, the reaction products of SBP1, H2 O2 , and H2 S also activated AMPK. CONCLUSIONS: Based on the obtained data, it is hypothesized that SBP1 negatively regulates oxidative phosphorylation (OXPHOS) in the healthy prostate cells by the production of H2 O2 and H2 S and consequential activation of AMPK. The reduction of SBP1 levels in prostate cancer can occur due to increased binding of HNF4α, acting as a transcriptional inhibitor to the SBP1 promoter. Consequently, there is a reduction in H2 O2 and H2 S-mediated signaling, inhibition of AMPK, and stimulation of OXPHOS and building blocks of biomolecules needed for tumor growth and progression. Other effects of SBP1 loss in tumor cells remain to be discovered.


Assuntos
Neoplasias da Próstata/metabolismo , Proteínas de Ligação a Selênio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Viral , Metilação de DNA , Progressão da Doença , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Masculino , Fosforilação Oxidativa , Consumo de Oxigênio , Células PC-3 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Quinases/metabolismo , Proteínas de Ligação a Selênio/deficiência , Proteínas de Ligação a Selênio/genética , Frações Subcelulares/metabolismo
15.
Prostate ; 80(12): 977-985, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32542727

RESUMO

BACKGROUND: Recently, resveratrol (Res) has been suggested to suppress the migration and invasion of prostate cancer (PCa). In the present study, we aimed to investigate the effects of Res on genomic DNA methylation, as well as the migration and invasion of PCa cells. METHODS: The suppression by Res of the growth of PCa cells was verified through a cytotoxicity assay. In addition, the effects of Res on 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and ten-eleven translocation 1 (TET1) levels were assessed, and the cell migration and invasion were also determined. The expressions of TET1, tissue inhibitor of metalloproteinases (TIMP) 2, TIMP3, MMP2, and MMP9 were detected through Western blot analysis. Afterward, TET1 was silenced using lentiviral short hairpin RNA to examine the effect of TET1 on the Res-triggered inhibition of migration and invasion of PCa cells. RESULTS: Our results showed that Res upregulated the 5hmC and TET1 levels and downregulated the 5mC level. Moreover, Res also inhibited the migration and invasion of PCa cells, promoted the demethylation of TIMP2 and TIMP3 to upregulate their expressions, and suppressed the expressions of MMP2 and MMP9. The silencing of TET1 in the presence of Res showed that Res could exert its effect through TET1. CONCLUSIONS: Our findings indicated that Res inhibited the migration and invasion of PCa cells via the TET1/TIMP2/TIMP3 pathway, which might potentially serve as a target for the treatment of PCa.


Assuntos
Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Resveratrol/farmacologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Invasividade Neoplásica , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Resveratrol/farmacocinética , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Inibidor Tecidual de Metaloproteinase-3/genética , Regulação para Cima
16.
Prostate ; 80(12): 986-992, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32557725

RESUMO

BACKGROUND: Focal therapies for prostate cancer (PC) can reduce adverse events and do not lead to androgen-independent progression. Ultrasound could be used for cancer treatments if the repetition frequency is fitted to the purpose. We investigated the possible therapeutic effect of ultrasound irradiation on PC cells. MATERIALS AND METHODS: We irradiated two PC cell lines, androgen-dependent LNCaP and -independent PC-3 with ultrasound (3.0 W/cm2 , 3 MHz, irradiation time rate: 20%) for 2 minutes for 1 day or 3 consecutive days at a repetition frequency of 1, 10, or 100 Hz in vitro. Cell proliferation and apoptosis were determined after irradiation. RESULTS: Cell proliferation of PC-3 was significantly inhibited after 1 day (P < .0001) and 3 days (P < .0001) of 10 Hz ultrasound irradiation, and that of LNCaP after 1 day (P < .0001) and 3 days (P < .0001) of irradiation. LNCaP was more sensitive to ultrasound at both lower and higher cell density but PC-3 was only sensitive at a lower cell density (P < .01). Irradiation with 10 Hz ultrasound-induced significantly more PC-3 apoptotic cells than control (1 day, P = .0137; 3 days, P = .0386) rather than irradiation with 1 Hz. Apoptosis via caspase-3 was induced at 10 Hz in 1-day (P < .05) irradiation in both cell lines. CONCLUSIONS: Ultrasound irradiation with even 1 day of 10 Hz significantly inhibited cell proliferation in both LNCaP and PC-3, especially by the remarkable induction of apoptosis in vitro. Our study indicated that ultrasound irradiation can be a therapeutic option for PC and further studies in vivo will be undertaken.


Assuntos
Neoplasias da Próstata/radioterapia , Terapia por Ultrassom/métodos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/radioterapia
17.
Life Sci ; 257: 117999, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585244

RESUMO

AIM: This paper was mainly aimed at synthesis of Ce-containing nano-Mg-phosphate ceramic as a multifunctional material. MATERIALS AND METHODS: Two ceramics based on Mg3(PO4)2 and Ce0.2Mg2.8(PO4)2 formulas (MP and MP-C, respectively) were synthesized. The synthesized powders were characterized by XRD, TEM, Zeta potential, and FTIR. Also, their dissolution behavior was tested in Tris-HCl buffer solution. Moreover, the antimicrobial efficacy was evaluated against gram-positive bacteria (Bacillus sphaericus MTCC 511 &Staphylococcus aureus MTCC 87) and gram-negative bacteria (Enterobacter aerogenes MTCC 111 &Pseudomonas aeruginosa MTCC 1034) using dick diffusion assay and microdilution method. Furthermore, the cell viability test was performed for the ceramics on Vero cells (African green monkey kidney cells), and their antitumor activity was determined by PC3 cell line (prostatic cancer). Also, the cellular uptake was determined by the flow cytometry. KEY FINDINGS: The results showed that the substitution of Mg by Ce decreased the particle size from 40 to 90 nm for MP sample to 2-10 nm for MP-C sample and increased the degradation rate. Both samples showed excellent antimicrobial activities. Moreover, MP demonstrated more cell viability than MP-C on Vero cells at high concentrations, whereas, MP-C showed more antitumor activity on PC3 cells than MP sample. Moreover, MP-C showed a higher cell uptake than MP due to its smaller size and more negative charge. SIGNIFICANCE: Mg-phosphate ceramic can be used in this study successfully as a delivery system for cerium ions and showed a high antitumor activity, which makes it highly recommended as safe and effective cancer treatment materials.


Assuntos
Cerâmica/farmacologia , Cério/farmacologia , Compostos de Magnésio/farmacologia , Fosfatos/farmacologia , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bacillaceae/efeitos dos fármacos , Osso e Ossos/microbiologia , Osso e Ossos/cirurgia , Sobrevivência Celular , Cério/metabolismo , Chlorocebus aethiops , Enterobacter aerogenes/efeitos dos fármacos , Humanos , Compostos de Magnésio/metabolismo , Testes de Sensibilidade Microbiana/métodos , Células PC-3 , Fosfatos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Células Vero
18.
Prostate ; 80(10): 753-763, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32421868

RESUMO

BACKGROUND: Although thrombospondins 4 (THBS4) participates in controlling the biology of prostate cancer (PCa), the mechanism underlying this regulation remains unknown. Hence, this study aims to identify the regulatory effects of THBS4 on the PCa stem cell-like properties and the potential mechanism associated with the phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (Akt) pathway. METHODS: PCa stem cells were sorted and identified using flow cytometry and THBS4 expression in the identified PCa stem cells was measured using Western blot assay. THBS4 was overexpressed or silenced in PCa stem cells, following which, self-renewal, proliferation, cell cycle distribution, and apoptosis of PCa stem cells were assessed as well as tumorigenicity in vivo was evaluated. PI3K/Akt pathway inhibitor was applied to identify its involvement in the regulatory roles of THBS4 in PCa stem cells. RESULTS: THBS4 was expressed at a higher level in PCa stem cells than in PCa cells. The overexpression of THBS4 promoted the self-renewal and proliferation, curbed the apoptosis of PCa stem cells, and enhanced the in vivo tumorigenicity, which was achieved by activating the PI3K/Akt pathway. On the contrary, short-hairpin RNA-mediated silencing of THBS4 exhibited suppressive effects on those cancer stem cell (CSC)-like properties and promotive effects on their apoptosis. CONCLUSION: THBS4 silencing can impede the CSC-like properties in PCa via blockade of the PI3K/Akt pathway, which provides patients with PCa a new therapeutic target.


Assuntos
Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombospondinas/metabolismo , Antígeno AC133/biossíntese , Animais , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células PC-3 , Neoplasias da Próstata/genética , Transdução de Sinais , Trombospondinas/biossíntese , Trombospondinas/deficiência , Trombospondinas/genética
19.
J Cancer Res Clin Oncol ; 146(8): 1953-1969, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32447485

RESUMO

OBJECTIVE: Prostate cancer (PCa) is an aggressive tumor. SHC SH2-domain-binding protein 1 (SHCBP1) has been identified frequently upregulated in various cancers, in addition to PCa. The aims of this study were to determine the relationships between SHCBP1 and clinicopathological characteristics of PCa and to explore the role of SHCBP1 in PCa proliferation and progression. METHODS: Tissue microarray and immunohistochemistry were used to determine the prognostic significance of SHCBP1. The relationship between clinicopathological characteristics of PCa and SHCBP1 was then analyzed using Cox regression analyses. To investigate SHCBP1 functions in vitro and in vivo, we knocked down SHCBP1 in PCa cell lines and established xenograft mice models. A series of cytological function assays were utilized to determine the role of SHCBP1 in cell proliferation, migration, invasion, and apoptosis. RESULTS: SHCBP1 was significantly upregulated in PCa tissues compared with BPH tissues. Patients with a higher expression of SHCBP1 were associated with poor survival outcomes than those with a lower expression of SHCBP1. Lentivirus-mediated shRNA knockdown of SHCBP1 in prostate cancer cell lines diminished cell growth, migration, and invasion dramatically both in vitro and in vivo, accompanied by an enhanced expression of large tumor suppressor 1 (LATS1) and tumor protein P53 (TP53) and inhibition of MDM2 proto-oncogene (MDM2), which suggested that SHCBP1 may promote proliferation and invasion in vitro via the LATS1-MDM2-TP53 pathway. The results of cycloheximide (CHX) and MG-132 assays indicated that SHCBP1 knockdown could attenuate the degradation of TP53 by the proteasome, prolong the half-life of TP53, and enhance the stabilization of TP53. CONCLUSION: These findings suggest that SHCBP1 overexpression contributes to PCa progression and that targeting SHCBP1 might be therapeutically beneficial to patients with PCa.


Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Bases de Dados Genéticas , Regulação para Baixo , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Células PC-3 , Prognóstico , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Adaptadoras da Sinalização Shc/biossíntese , Proteínas Adaptadoras da Sinalização Shc/genética , Análise Serial de Tecidos , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
20.
Toxicol Appl Pharmacol ; 401: 115071, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454055

RESUMO

Prostate Cancer (PCa) is the second most common cancer among men in United States after skin cancer. Conventional chemotherapeutic drugs available for PCa treatment are limited due to toxicity and resistance issues. Therefore, there is an urgent need to develop more effective treatment for advanced PCa. In this current study, we focused on evaluating the anti-cancer efficacy of Eprinomectin (EP), a novel avermectin analog against PC3 metastatic PCa cells. EP displayed robust inhibition of cell viability of PC3 cells in addition to suppressing the colony formation and wound healing capabilities. Our study showed that EP targets PC3 cells via inducing ROS and apoptosis activation. EP treatment enforces cell cycle arrest at G0/G1 phase via targeting cyclin-dependent kinase 4 (CDK4) and subsequent induction of apoptosis in PC3 cells. At the molecular level, EP effectively inhibited the expression of various cancer stem cell markers such as ALDH1, Sox-2, Nanog, Oct3/4 and CD44. Interestingly, EP also inhibited the activity of alkaline phosphatase, a maker of pluripotent stem cells. Of note, EP treatment resulted in the translocation of ß-catenin from the nucleus to the cytoplasm indicating that EP antagonizes Wnt/ß-catenin signaling pathway. Western blotting analysis revealed that EP downregulated the expression of key cell cycle markers such as cyclin D1, cyclin D3, CDK4, and c-Myc. In addition, EP inhibited the anti-apoptotic markers such as Mcl-1, XIAP, c-IAP1 and survivin in PC3 cells. On the other hand, EP treatment resulted in the activation of pH2A.X, Bad, caspase-9, caspase-3 and cleavage of PARP1. Taken together, our data suggests that EP is a potential agent to treat advanced PCa cells via modulating apoptosis signaling.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ivermectina/análogos & derivados , Lactonas/farmacologia , Compostos Macrocíclicos/farmacologia , Neoplasias da Próstata/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Citotoxinas/química , Citotoxinas/farmacologia , Citotoxinas/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Ivermectina/química , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Lactonas/uso terapêutico , Compostos Macrocíclicos/química , Compostos Macrocíclicos/uso terapêutico , Masculino , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
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