RESUMO
INTRODUCTION: Regucalcin plays a multifunctional role in the regulation of cellular function including metabolism, signaling process, and transcriptional activity in maintaining cell homeostasis. Downregulated expression or activity of regucalcin contributes to the development of malignancies in various types of human cancer. Survival of cancer patients, including metastatic prostate cancer, is prolonged with high expression of regucalcin in the tumor tissues. METHODS: We elucidate whether extracellular regucalcin conquers the growth, migration, invasion, and adhesion of metastatic human prostate cancer PC-3 and DU-145 cells. RESULTS: Extracellular regucalcin (0.1, 1, and 10 nM) of physiologic levels (1 nM at human serum) inhibited colony formation and growth of PC-3 and DU-145 cells, while it did not have an effect on cell death. Repressive effects of extracellular regucalcin on the proliferation were not exhibited by the presence of inhibitors of the cell cycle, intracellular signaling process, and transcriptional activity, suggesting that the signals of extracellular regucalcin are transmitted to block cell growth. Furthermore, extracellular regucalcin (0.1, 1, or 10 nM) inhibited migration, invasion, and adhesion of PC-3 and DU-145 cells. Mechanistically, extracellular regucalcin (10 nM) decreased the levels of various signaling proteins including Ras, posphatidylinositol-3 kinase, mitogen-activated protein kinase, mechanistic target of rapamycin, RSK-2, caveolin-1, and integrin ß1 in PC-3 cells. DISCUSSION AND CONCLUSION: Thus, extracellular regucalcin may play a suppressive role in growth, migration, invasion, and adhesion, which are involved in the metastatic activity of human prostate cancer cells, via affecting diverse signaling processes. This study may provide a new strategy in preventing metastatic prostate cancer with exogenous regucalcin.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias da Próstata , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , MasculinoRESUMO
In recent studies, it has been reported that ion channels play an important role in cancer formation. Therefore, it is possible that the use of pharmacological agents targeting ion channels will allow the development of new strategies for cancer treatment. In this study, we investigate the effect of imipramine on Eag1 channel expression in DU145 prostate cancer cells. Culture cells were divided into 4 groups as the control, 10, 50 and 75 µM imipramine. Eag1 channel currents and conductivity were determined by whole-cell patch-clamp technique and gene expression by real time-polymerase chain reaction (RT-PCR). Current records were taken before (at 0th minute, as control) and 10 min after imipramine administration to the cells. It was observed that all three doses of imipramine significantly reduced Eag1 currents and conductivity compared with the control. However, the differences between dose groups were not significant. Similarly, Eag1 channel protein expression was found to be significantly reduced for all three doses of imipramine compared with the control group, but there was no significant difference in gene expression between dose groups. Obtained results suggested that imipramine has the potential to be used as a pharmacological agent targeting the Eag1 channel in the treatment of prostate cancer.
Assuntos
Imipramina , Neoplasias da Próstata , Linhagem Celular Tumoral , Éter , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Imipramina/farmacologia , Masculino , Neoplasias da Próstata/tratamento farmacológicoRESUMO
In previous work, we showed that highly proliferative cells and cancer cells, but not cells with normal growth rate, have tubules rich in alpha-1,2 fucosylated epitopes that extend radially from the nucleus to the cell periphery and form an unusual uptake system. The importance of alpha-1,2 fucosylation in forming tubules was demonstrated by proving that down-regulating the two corresponding fucosyltransferases (FUT1 and FUT2) causes tubule fragmentation. Here, we present evidence that in the prostate cancer cell line DU145, the tubules arise in actively growing cells from vesicles in the medial and trans elements of a partially fragmented Golgi complex, while in not actively growing cells the tubules become completely independent from the Golgi complex. Formation and elongation of the tubules proved to depend on the actin cytoskeleton, since the alpha-1,2 fucosylated protein(s) segregate with the cytoskeleton proteins, and not in the membrane fraction, as do the Golgi markers and other fucosylated proteins, while depolymerization of the actin filaments causes tubule fragmentation and shifting of the alpha-1,2 fucosylated proteins into the membrane fraction.
Assuntos
Actinas/metabolismo , Fucose/metabolismo , Complexo de Golgi/metabolismo , Neoplasias da Próstata/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Citocalasinas/farmacologia , Epitopos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Humanos , Lectinas/metabolismo , Masculino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/ultraestruturaRESUMO
Relatively little is known about the endocannabinoid system in human neuroblastoma cell lines. In the present study, we have investigated the expression of the genes coding for the enzymes involved in the synthesis and catabolism of endocannabinoids in the SH-SY5Y cell line. The expression of MGLL, the gene coding for the 2-arachidonoylglycerol hydrolytic enzyme monoacylglycerol lipase (MAGL), was found to be 85 and 340 fold lower than the expression levels for the genes coding for alpha/beta-hydrolase domain containing 6 and 12 (ABHD6, ABHD12), which are alternative hydrolytic enzymes for this endocannabinoid. In comparison, mRNA levels of MGLL were 1.5 fold higher than ABHD6 and 2 fold lower than the levels of ABHD12 in DU-145 human prostate cells. In functional assays, the hydrolysis of the 2-arachidonoylglycerol homologue 2-oleoylglycerol by intact SH-SY5Y cells was partially inhibited by the ABHD6 inhibitor WWL70, but not by the MAGL inhibitor JZL184, whereas the reverse was true in DU-145 cells. The combination of JZL184 + WWL70 did, however produce a significantly greater inhibition of 2-OG hydrolysis than seen with WWL70 alone in the SH-SY5Y cells. The low MGLL expression in the SH-SY5Y cells was not due to epigenetic silencing, since levels were not affected by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone acetylase inhibitor trichostatin A. The low MGLL expression in SH-SY5Y cells should be taken into account when using these cells in experiments investigating the involvement of the endocannabinoid system in models of physiological and pathological processes.
Assuntos
Regulação Neoplásica da Expressão Gênica , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Neuroblastoma/patologia , Linhagem Celular Tumoral , Decitabina/farmacologia , Endocanabinoides/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicerol/metabolismo , Humanos , Hidrólise , Ácidos Hidroxâmicos/farmacologia , RNA Mensageiro/genéticaRESUMO
Ouabain, a cardiotonic steroid and specific Na+ /K+ -ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca2+ , and mitochondrial membrane potential (ΔΨm ) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca2+ production, but decreased the levels of ΔΨm in DU 145 cells. Ouabain also increased the activities of caspase-3, -8, and -9. Western blotting was used for measuring the alterations of apoptosis-associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATMSer1981 , p-H2A.XSer139 , and p-p53Ser15 ) and ER-stress-associated proteins (Grp78, ATF6ß, p-PERKThr981 , PERK, eIF2A, GADD153, CaMKIIß, and caspase-4) in time-dependently. Furthermore, ouabain increased apoptosis-associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro-apoptotic protein Bax, increased apoptotic-associated proteins, such as cytochrome c, AIF, Endo G, caspase-3, -8, and -9, but reduced anti-apoptotic protein Bcl-2 and Bcl-x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase-dependent and mitochondria-dependent pathways in human prostate cancer DU 145 cells.
Assuntos
Apoptose/efeitos dos fármacos , Cardiotônicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Ouabaína/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
We investigated the antitumor mechanism of Anthopleura anjunae oligopeptide (AAP-H, YVPGP) in prostate cancer DU-145 cells in vitro and in vivo. Results indicated that AAP-H was nontoxic and exhibited antitumor activities. Cell cycle analysis indicated that AAP-H may arrest DU-145 cells in the S phase. The role of the phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein (PI3K/AKT/mTOR) signaling pathway in the antitumor mechanism of APP-H was investigated. Results showed that AAP-H treatment led to dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered compared to the untreated DU-145 cells. Inhibition of PI3K/AKT/mTOR signaling pathway in the DU-145 cells by employing inhibitor LY294002 (10 µM) or rapamycin (20 nM) effectively attenuated AAP-H-induced phosphorylation of AKT and mTOR. At the same time, inhibitor addition further elevated AAP-H-induced cleaved-caspase-3 levels. Furthermore, the effect of AAP-H on tumor growth and the role of the PI3K/AKT/mTOR signaling pathway in nude mouse model were also investigated. Immunohistochemical analysis showed that activated AKT, PI3K, and mTOR levels were reduced in DU-145 xenografts. Western blotting showed that AAP-H treatment resulted in dose-dependent reduction in p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) levels, whereas t-AKT and t-PI3K levels remained unaltered. Similarly, Bcl-xL levels decreased, whereas that of Bax increased after AAP-H treatment. AAP-H also increased initiator (caspase 8 and 9) and executor caspase (caspase 3 and 7) levels. Therefore, the antitumor mechanism of APP-H on DU-145 cells may involve regulation of the PI3K/AKT/mTOR signaling pathway, which eventually promotes apoptosis via mitochondrial and death receptor pathways. Thus, the hydrophobic oligopeptide (YVPGP) can be developed as an adjuvant for the prevention or treatment of prostate cancer in the future.
Assuntos
Proliferação de Células/efeitos dos fármacos , Oligopeptídeos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Anêmonas-do-Mar , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Morfolinas/farmacologia , Células NIH 3T3 , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, belonging to the Vitaceae family and Vitis genus, and widely used as folk herbal medicine. It is traditionally used for the treatment of diarrhea, hypertension, neuroprotection, jaundice, and arthritis. We used the wild-collected VTT and sterilized them to establish the plant tissue culture, and then took the leaves for DNA sequencing to determine its original base. We use methanol to extract VTT in four different solvents: 1-butanol, n-hexane, ethyl acetate, and water. These four preliminary extracts were used to treat human prostate cancer DU145 cells in vitro. We use the flow cytometry to check the cell survival situation. Finally, we found the ethyl acetate layer roughing product (referred VTEA) in human prostate cancer apoptotic effects of cell line DU-145. In the present studies, we use the crude extract of VTT to examine whether or not it can induce apoptosis of DU145 cells in vitro. Viability assays for extracts of VTT treatment showed that it had dose-dependent effect on human prostate cancer DU145 cells. We also found that the extract of VTT induces time-dependent mitochondrial and intrinsic-dependent apoptosis pathways. The in vitro cytotoxic effects were investigated by cell cycle analysis and the determination of apoptotic DNA fragmentation in DU145 cells. The cell cycle analysis showed that extracts of VTT induced a significant increase in the number of cells in G0 /G1 phase. The extract of VTT induced chromatin changes and apoptosis of DU145 cells also were confirmed by DAPI and PI staining that were measured by fluorescence microscopy and flow cytometry, respectively. Finally, the expression of relevant proteins was analyzed by Western blot analysis. These results promoted us to further evaluate apoptosis associated proteins and elucidate the possible signal pathway in DU-145 cells after treated with the extract of VTT.
Assuntos
Apoptose/efeitos dos fármacos , Ciclina D/metabolismo , Ciclina E/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vitis/química , Acetatos/química , Caspases/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ciclina D/antagonistas & inibidores , Ciclina E/antagonistas & inibidores , Fragmentação do DNA/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metanol/química , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Extratos Vegetais/análise , Extratos Vegetais/química , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio/metabolismo , Taiwan , Vitis/metabolismoRESUMO
BACKGROUND: Present study aimed to better understand the potential apoptotic pathways that involved in docosahexaenoic acid (DHA)-induced apoptosis of prostate cancer cells. METHODS: Human prostate cancer DU145 cells were treated with different concentrations of fish oil, omega-3 PUFA (DHA, and Eicosapentaenoic acid, EPA), or omega-6 PUFA (Arachidonic acid, AA). Cell viability and apoptosis were evaluated by MTT assay and Hoechst staining. Pathway-focused gene expression profiling of DU145 cells was analyzed with the RT2 Profile PCR Array System. The results were verified by real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: AA exposure showed no obvious effect on viability of DU145 cells. However, exposure with fish oil, EPA, or DHA for 24 h significantly affected cell viability. The growth inhibition of DHA was more pronounced than that of EPA and showed a time-dependent increase. DHA exposure caused typical apoptotic characteristics. Ten genes were more expressed, while 5 genes were less expressed following DHA exposure. RT-qPCR confirmed the time dependent effect of DHA on the expression of these differentially expressed genes. KEGG pathway analysis showed that DHA may induce the apoptosis of cancer cells preferentially through mediating P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. CONCLUSION: Our study demonstrated the beneficial action of DHA on human prostate carcinoma cell line DU145. The pro-apoptotic effect of DHA on DU145 cells may involve mediation various pathways, especially P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Molecular mechanisms of DHA on apoptosis of cancer cells still need to be further clarified.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Regulação Neoplásica da Expressão Gênica , Próstata/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Próstata/metabolismo , Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In this study, the antitumor activity of KHC-4 was analyzed using human prostate cancer (CaP) cells and the underlining anticancer mechanisms of KHC-4 were identified. KHC-4 inhibited cell proliferation and induced cytotoxicity in the castration-resistant CaP DU145 cell line. The most effective concentration of KHC-4 was 0.1 µM. Cell cycle analysis demonstrated that KHC-4 treatment caused G2/M arrest and a subsequent increase in the sub-G1 population. Furthermore, KHC-4 is up-regulated p21, p27, and p53 in a time- and concentration-dependent manner. The exposure of cells to KHC-4 induced Cdk1/cyclin B1 complex activity, which led to cell cycle arrest. Moreover, KHC-4 inhibited the activities of MMP-2 and MMP-9 to inhibit tumor cell metastasis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1879-1887, 2016.
Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Morfolinas/farmacologia , Quinolonas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração , Regulação para CimaRESUMO
At the molecular level, several developmental signaling pathways, such as Wnt/ß-catenin, have been associated with the initiation and subsequent progression of prostate carcinomas. The present report elucidated the anti-cancerous attributes of an anthraquinone, aloe-emodin (AE), against androgen-independent human prostate cancer DU145 cells. The cytotoxicity profiling of AE showed that it exerted significant cytotoxic effects and increased lactose dehydrogenase levels in DU145 cells (p < 0.01 and p < 0.001). AE also induced considerable reactive oxygen species (ROS)-mediated oxidative stress, which escalated at higher AE concentrations of 20 and 25 µM. AE also efficiently instigated nuclear fragmentation and condensation concomitantly, followed by the activation of caspase-3 and -9 within DU145 cells. AE further reduced the viability of mitochondria with increased cytosolic cytochrome-c levels (p < 0.01 and p < 0.001) in DU145 cells. Importantly, AE exposure was also correlated with reduced Wnt2 and ß-catenin mRNA levels along with their target genes, including cyclin D1 and c-myc. Furthermore, the molecular mechanism of AE was evaluated by performing molecular docking studies with Wnt2 and ß-catenin. Evidently, AE exhibited good binding energy scores toward Wnt2 and ß-catenin comparable with their respective standards, CCT036477 (Wnt2 inhibitor) and FH535 (ß-catenin inhibitor). Thus, it may be considered that AE was competent in exerting anti-growth effects against DU145 androgen-independent prostate cancer cells plausibly by modulating the expression of Wnt/ß-catenin signaling.
RESUMO
Calcium signaling controls a large variety of cell functions, including proliferation and apoptosis, and plays a major role in neoplastic transformation. Prostate cancer (PCa) is one of the most common malignancies in men. The transition to castration-resistant prostate cancer (CRPC), a lethal form that is still lacking an effective cure, could be influenced by fine tuning intracellular calcium ([Ca2+]i) homeostasis. This study investigates [Ca2+]i dynamics in metastatic PCa cell lines that mimic the progression of PCa to CRPC: (i) well differentiated LNCaP cells that require androgen for survival, and (ii) poorly differentiated, highly aggressive androgen-insensitive prostate cancer (AIPC) PC3 and DU145 cells. In AIPC cells, ATP induces a fast rise in [Ca2+]i, due to release from intracellular stores and sensitive to phospholipase C inhibitors, while LNCaP cells do not respond to ATP challenge. Moreover, AIPC cells showed a reduced capacity to store Ca2+ in thapsigargin-sensitive stores and limited store-operated calcium entry, with respect to androgen-dependent LNCaP cells. Finally, green tea extract causes [Ca2+]i elevation and inhibits proliferation in PC3 and DU145 cells, but is ineffective in LNCaP cells. The consequences of these differences are discussed and interpreted in this study with reference to previously proposed models for Ca2+ dependence of prostate carcinogenesis.
Assuntos
Próstata , Neoplasias de Próstata Resistentes à Castração , Androgênios , Sinalização do Cálcio , Linhagem Celular Tumoral , Humanos , Masculino , Próstata/metabolismo , Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
To model the problem of radiation resistance in prostate cancer, cell lines mimicking a clinical course of conventionally fractionated or hypofractionated radiotherapy have been generated. Proteomic analysis of radiation resistant and radiosensitive DU145 prostate cancer cells detected 4410 proteins. Over 400 proteins were differentially expressed across both radiation resistant cell lines and pathway analysis revealed enrichment in epithelial to mesenchymal transition, glycolysis and hypoxia. From the radiation resistant protein candidates, the cell surface protein CD44 was identified in the glycolysis and epithelial to mesenchymal transition pathways and may serve as a potential therapeutic target.
Assuntos
ProteômicaRESUMO
Despite advances in treatment of lethal prostate cancer, the incidence of prostate cancer brain metastases is increasing. In this sense, we analyzed the molecular profile, as well as the functional consequences involved in the reciprocal interactions between prostate tumor cells and human astrocytes. We observed that the DU145 cells, but not the LNCaP cells or the RWPE-1 cells, exhibited more pronounced, malignant and invasive phenotypes along their interactions with astrocytes. Moreover, global gene expression analysis revealed several genes that were differently expressed in our co-culture models with the overexpression of GLIPR1 and SPARC potentially representing a molecular signature associated with the invasion of central nervous system by prostate malignant cells. Further, these results were corroborated by immunohistochemistry and in silico analysis. Thus, we conjecture that the data here presented may increase the knowledge about the molecular mechanisms associated with the invasion of CNS by prostate malignant cells.
Assuntos
Neoplasias Encefálicas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Neoplasias da Próstata/genética , Células A549 , Animais , Astrócitos/química , Astrócitos/citologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Regulação para CimaRESUMO
It has been reported that B-type procyanidins can alleviate oxidative damage of prostatic cells, but there has been limited information on the similar role of A-type procyanidins. This study investigated the protective effect of procyanidin A-type dimers from peanut skin against H2O2-induced oxidative stress damage in prostate cancer DU145 cells. According to the UPLC-Q-TOF-MS/MS analysis and comparison with standards, the fourth fraction of peanut skin procyanidin (PSP-4) was identified as procyanidin A-type dimers, namely, procyanidin A1 and A2. Results revealed that PSP-4 treatment prior H2O2 exposure increased cell activity and attenuated the cell cycle arrest and apoptosis rate. The H2O2-induced increase in intracellular reactive oxygen species (ROS) was remarkably inhibited by PSP-4. PSP-4 treatment enhanced the activity of catalase (CAT) and total super oxide dismutase (T-SOD) and restored glutathione (GSH) content, compared with the H2O2 treatment. Furthermore, the results indicated that PSP-4 protected DU145 cells by attenuating phosphorylation of the mitogen-activated protein kinases (MAPKs), by increasing the Bcl-2/Bax ratio, and by reducing the activation of caspase-3 and caspase-9 by cascade reactions. This study reveals that procyanidin A-type dimers from peanut skin have the potential function in preventing oxidative stress damage of prostatic cells.
Assuntos
Catequina/farmacologia , Peróxido de Hidrogênio/efeitos adversos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxidantes/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Proantocianidinas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Substâncias Protetoras/farmacologia , Apoptose , Arachis/química , Ciclo Celular , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Long noncoding RNAs (lncRNAs) are involved in the pathogenesis of prostate cancer (PCa) as competitive endogenous RNA. The present study aimed to investigate the molecular mech--anisms of lncRNA small nucleolar RNA host gene 16 (SNHG16) in the proliferation and metastasis of PCa cells. Cancer tissues and adjacent normal tissues were collected from 80 patients with PCa who did not receive any treatment. Reverse transcriptionquantitative PCR analysis was performed to detect the expression levels of SNHG16, hsamicroRNA (miRNA/miR)3733p and transforming growth factorß receptor type 2 (TGFßR2), and Spearman's correlation coefficient analysis was performed to assess the correlations between these molecules. Furthermore, the effects of SNHG16 knockdown and overexpression on the biological functions of DU145 PCa cells and TGFßR2/SMAD signaling were analyzed. The dualluciferase reporter assay was performed to assess the associations between SNHG16 and miR3733p, and TGFßR2 and miR3733p, the effects of which were verified via rescue experiments. The results demonstrated that the expression levels of SNHG16 and TGFßR2 were significantly upregulated in PCa tissues, whereas miR3733p expression was significantly downregulated (P<0.001). In addition, negative correlations were observed between SNHG16 and miR3733p (rho, 0.631) and miR3733p and TGFßR2 (rho, 0.516). Overexpression of SNHG16 significantly promoted the proliferation, migration and invasion of PCa cells (P<0.05), and significantly increased the protein expression levels of TGFßR2, phosphorylated (p)SMAD2, pSMAD3, cMyc and E2F4 (P<0.001). Notably, the results revealed that miR3733p is a target of SNHG16, and miR3733p knockdown rescued short hairpin (sh)SNHG16suppressed cellular functions by promoting TGFßR2/SMAD signaling. The results also revealed that miR3733p targets TGFßR2. Notably, transfection with miR3733p inhibitor rescued shTGFßR2suppressed cell proliferation and migration. Taken together, the results of the present study suggest that SNHG16 promotes the proliferation and migration of PCa cells by targeting the miR3733p/TGFßR2/SMAD axis.
Assuntos
MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Longo não Codificante/genética , Fator de Crescimento Transformador beta/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Regulação para CimaRESUMO
Many studies have shown that pollen and its preparation are ideal herbal remedies for the treatment of prostate diseases. Our previous study found that pollen polysaccharides from Chinese wolfberry (WPPs) can induce the apoptosis of prostate cancer DU145 cells. But the antitumor mechanism of WPPs was not clearly understood. Therefore, in the present study, we further investigated the antitumor mechanism of WPPs in DU145 cells and a xenograft mice model. The results showed that WPPs decreased the levels of PI3K, AKT, p-AKT and Bcl-2 proteins, and increased expression of Bax, caspase-3 and caspase-9 in DU145 cells (P < 0.05). The in vivo data demonstrated that WPPs resulted in a significant dose-dependent increase (P < 0.05) in the number of apoptotic cells in tumor tissues. Immunohistochemical analysis showed that the activated PI3K, AKT, p-AKT and Bcl-2 levels were decreased and the level of caspase-3 was increased in DU145 xenografts mice model. Therefore, the antitumor mechanism of WPPs on DU145 cells may involve regulation of the PI3K/AKT signaling pathway, which eventually promotes apoptosis. This study provided the experimental basis for further studied of WPPs as a possible functional food or adjuvant agent for prevention or treatment of prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Lycium/química , Fosfatidilinositol 3-Quinases/metabolismo , Pólen/química , Polissacarídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , China , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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-3RESUMO
Casticin, a polymethoxyflavone derived from natural plants, has biological activities including induction of cell apoptosis. In this study, we showed the beneficial effects of casticin on the inhibition of prostate cancer cell metastasis. Casticin reduced total viable cell number, thus, we selected low doses of casticin for following experiments. Casticin decreased cell mobility, suppressed cell migration and invasion, and reduced cell gelatinolytic activities of MMP-2/-9. Furthermore, casticin inhibited the protein levels of AKT, GSK3 αß, Snail, and MMPs (MMP-2, -9, -13, and -7) at 24 and 48 hr treatment. Casticin diminished the expressions of NF-κB p65, GRB2, SOS-1, MEK, p-ERK1/2, and p-JNK1/2 at 48 hr treatment only. However, casticin reduced the level of E-cadherin at 24 hr treatment but elevated at 48 hr. The novel findings suggest that casticin may represent a new and promising therapeutic agent for the metastatic prostate cancer. PRACTICAL APPLICATIONS: Casticin derived from natural plants had been used for Chinese medicine in Chinese population for thousands of years. In the present study, casticin attenuated metastatic effects, including decreasing viable cell number, inhibiting the migration, invasion, and adhesion, and reducing matrix metalloproteinases activity on human prostate DU 145 cancer cells. In addition, the results also provided possible pathways involved in casticin anti-metastasis mechanism. We conclude that casticin may be an aptitude anticancer agent or adjuvant for the metastatic prostate cancer in the future.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Flavonoides/farmacologia , Proteína Oncogênica v-akt/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Flavonoides/química , Humanos , Masculino , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Fator de Transcrição RelA/metabolismo , Proteínas ras/metabolismoRESUMO
BACKGROUND/AIM: Identification of prostatic stem cells in primary prostate tissue sections, organ cultures of prostate and cell lines requires a range of techniques that allows characterization of stem cells for their potential use in the treatment of patients. Isolated cells usually round-up and develop changes in shape, size and cellular characteristics. The aim of this study was to provide a range of methods for identifying prostatic stem cells and characterizing them with regard to ultrastructure, nuclear morphology, cytoplasmic organelles, and/or expression stem cell marker CD133. MATERIALS AND METHODS: Prostate biopsy and prostatectomy specimens were used for studying prostatic stem cells and their known marker CD133 in tissue sections by light and/or electron microscopy. Inverted capsule embedding was used to study archival metastatic prostate in pelvic nodes and Du145 cell line in a monolayer culture. RESULTS: Staining for CD133 positively identified stem cells that were found in benign prostatic hyperplasia, benign prostate, and prostate cancer cells. Paraffin embedded sections showed a single type of stem cells, whereas methylene blue-stained Epon sections showed both light and dark stem cells. Electron microscopy showed that both basal and stem cells were closely associated with the basement membrane (basal lamina). Stem cells had smooth plasma and nuclear membranes, a prominent nucleolus, small mitochondria, and few ribosomes. Du145 cells were separated by intercellular spaces in monolayer culture. CONCLUSION: The inverted capsule embedding method allowed the study of metastasized prostate cancer in pelvic lymph nodes. Our approach enabled the assessment of stem cells in tissue sections by light and electron microscopy.
Assuntos
Antígeno AC133/genética , Membrana Basal/ultraestrutura , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Membrana Basal/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Microscopia Eletrônica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/ultraestrutura , Próstata/metabolismo , Próstata/patologia , Próstata/ultraestrutura , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/ultraestruturaRESUMO
The dynamic and never exactly repeatable tumor transcriptomic profile of people affected by the same form of cancer requires a personalized and time-sensitive approach of the gene therapy. The Gene Master Regulators (GMRs) were defined as genes whose highly controlled expression by the homeostatic mechanisms commands the cell phenotype by modulating major functional pathways through expression correlation with their genes. The Gene Commanding Height (GCH), a measure that combines the expression control and expression correlation with all other genes, is used to establish the gene hierarchy in each cell phenotype. We developed the experimental protocol, the mathematical algorithm and the computer software to identify the GMRs from transcriptomic data in surgically removed tumors, biopsies or blood from cancer patients. The GMR approach is illustrated with applications to our microarray data on human kidney, thyroid and prostate cancer samples, and on thyroid, prostate and blood cancer cell lines. We proved experimentally that each patient has his/her own GMRs, that cancer nuclei and surrounding normal tissue are governed by different GMRs, and that manipulating the expression has larger consequences for genes with higher GCH. Therefore, we launch the hypothesis that silencing the GMR may selectively kill the cancer cells from a tissue.