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1.
Theranostics ; 9(9): 2526-2540, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131051

RESUMO

Rationale: Cancer cells reprogram cellular metabolism to fulfill their needs for rapid growth and metastasis. However, the mechanism controlling this reprogramming is poorly understood. We searched for upregulated signaling in metastatic colorectal cancer and investigated the mechanism by which Glut3 promotes tumor metastasis. Methods: We compared RNA levels and glycolytic capacity in primary and metastatic colon cancer. The expression and association of Glut3 with clinical prognosis in colon cancer tissues was determined by immunohistochemistry. Glut3 gain-of-function and loss-of-function were established using colon cancer HCT116, HT29, and metastatic 116-LM cells, and tumor invasiveness and stemness properties were evaluated. Metabolomic profiles were analyzed by GC/MS and CE-TOF/MS. The metastatic burden in mice fed a high-fat sucrose diet was assessed by intravenous inoculation with Glut3 knockdown 116-LM cells. Results: Upregulation of glycolytic genes and glycolytic capacity was detected in metastatic colorectal cancer cells. Specifically, Glut3 overexpression was associated with metastasis and poor survival in colorectal cancer patients. Mechanistically, Glut3 promoted invasiveness and stemness in a Yes-associated protein (YAP)-dependent manner. Activation of YAP in turn transactivated Glut3 and regulated a group of glycolytic genes. Interestingly, the expression and phosphorylation of PKM2 were concomitantly upregulated in metastatic colorectal cancer, and it was found to interact with YAP and enhance the expression of Glut3. Importantly, a high-fat high-sucrose diet promoted tumor metastasis, whereas the inhibition of either Glut3 or YAP effectively reduced the metastatic burden. Conclusion: Activation of the Glut3-YAP signaling pathway acts as a master activator to reprogram cancer metabolism and thereby promotes metastasis. Our findings reveal the importance of metabolic reprogramming in supporting cancer metastasis as well as possible therapeutic targets.

2.
Int J Oncol ; 54(5): 1639-1650, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896828

RESUMO

Reactive oxygen species (ROS) homeostasis is maintained at a higher level in cancer cells, which promotes tumorigenesis. Oxidative stress induced by anticancer drugs may further increase ROS to promote apoptosis, but can also enhance the metastasis of cancer cells. The effects of ROS homeostasis on cancer cells remain to be fully elucidated. In the present study, the effect of a reduction in manganese superoxide dismutase (MnSOD) on the migration and invasion of A431 cells was investigated. Our previous micro­assay data revealed that the mRNA expression of MnSOD was higher in the invasive A431­III cell line compared with that in the parental A431 cell line (A431­P). In the present study, high protein levels of MnSOD and H2O2 production were observed in A431­III cells; however, catalase protein levels were significantly lower in A431­III cells compared with those in the A431­P cell line. The knockdown of MnSOD increased H2O2 levels, enzyme activity, the mRNA levels of matrix metalloproteinase­1, ­2 and ­9, and the migratory and invasive abilities of the cells. Inducing a reduction in H2O2 using diphenyleneiodonium (DPI) and N­acetyl­l­cysteine decreased the migratory abilities of the cell lines, and DPI attenuated the migratory ability that had been increased by MnSOD small interfering RNA knockdown. Luteolin (Lu) and quercetin (Qu) increased the expression of catalase and reduced H2O2 levels, but without an observed change in the protein levels of MnSOD. Taken together, these data suggest that reduced MnSOD may induce ROS imbalance in cells and promote the metastatic ability of cancer cells. Lu and Qu may attenuate these processes and may be promising potential anticancer agents.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação para Baixo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Acetilcisteína/farmacologia , Carcinoma de Células Escamosas/metabolismo , Movimento Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Luteolina/farmacologia , Metaloproteinases da Matriz/genética , Invasividade Neoplásica , Oniocompostos/farmacologia , Quercetina/farmacologia
3.
Oncogene ; 2018 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30072740

RESUMO

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8's oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8's functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

4.
J Food Drug Anal ; 26(3): 1180-1191, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29976410

RESUMO

Flavonoids luteolin and quercetin can inhibit growth and metastasis of cancer cells. In our previous report, luteolin and quercetin was shown to block Akt/mTOR/c-Myc signaling. Here, we found luteolin and quercetin reduced protein level and transactivation activity of RPS19 in A431-III cells, which is isolated from parental A431 (A431-P) cell line. Further investigation the inhibitory mechanism of luteolin and quercetin on RPS19, we found c-Myc binding sites on RPS19 promoter. The Akt inhibitor LY294002, mTOR inhibitor rapamycin and c-Myc inhibitor 10058-F4 significantly suppressed RPS19 expression and transactivation activities. Overexpression and knockdown of c-Myc in cancer cells show RPS19 expression was regulated by c-Myc. Furthermore, Knockdown and overexpression of RPS19 was used to analyze of the function of RPS19 in cancer cells. The epithelial-mesenchymal transition (EMT) markers and metastasis abilities of cancer cells were also regulated by RPS19. These data suggest that luteolin and quercetin might inhibit metastasis of cancer cells by blocking Akt/mTOR/c-Myc signaling pathway to suppress RPS19-activated EMT signaling.

5.
Clin Cancer Res ; 24(5): 1176-1189, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29222162

RESUMO

Purpose: MPT0L145 has been developed as a FGFR inhibitor exhibiting significant anti-bladder cancer activity in vitro and in vivo via promoting autophagy-dependent cell death. Here, we aim to elucidate the underlying mechanisms.Experimental Design: Autophagy flux, morphology, and intracellular organelles were evaluated by Western blotting, transmission electron microscope, and fluorescence microscope. Molecular docking and surface plasmon resonance assay were performed to identify drug-protein interaction. Lentiviral delivery of cDNA or shRNA and CRISPR/Cas9-mediated genome editing was used to modulate gene expression. Mitochondrial oxygen consumption rate was measured by a Seahorse XFe24 extracellular flux analyzer, and ROS level was measured by flow cytometry.Results: MPT0L145 persistently increased incomplete autophagy and phase-lucent vacuoles at the perinuclear region, which were identified as enlarged and alkalinized late-endosomes. Screening of a panel of lipid kinases revealed that MPT0L145 strongly inhibits PIK3C3 with a Kd value of 0.53 nmol/L. Ectopic expression of PIK3C3 reversed MPT0L145-increased cell death and incomplete autophagy. Four residues (Y670, F684, I760, D761) at the ATP-binding site of PIK3C3 are important for the binding of MPT0L145. In addition, MPT0L145 promotes mitochondrial dysfunction, ROS production, and DNA damage, which may in part, contribute to cell death. ATG5-knockout rescued MPT0L145-induced cell death, suggesting simultaneous induction of autophagy is crucial to its anticancer activity. Finally, our data demonstrated that MPT0L145 is able to overcome cisplatin resistance in bladder cancer cells.Conclusions: MPT0L145 is a first-in-class PIK3C3/FGFR inhibitor, providing an innovative strategy to design new compounds that increase autophagy, but simultaneously perturb its process to promote bladder cancer cell death. Clin Cancer Res; 24(5); 1176-89. ©2017 AACR.

6.
Nat Commun ; 8: 15874, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28639619

RESUMO

Long noncoding RNAs (lncRNAs) have been implicated in hypoxia/HIF-1-associated cancer progression through largely unknown mechanisms. Here we identify MIR31HG as a hypoxia-inducible lncRNA and therefore we name it LncHIFCAR (long noncoding HIF-1α co-activating RNA); we describe its oncogenic role as a HIF-1α co-activator that regulates the HIF-1 transcriptional network, crucial for cancer development. Extensive analyses of clinical data indicate LncHIFCAR level is substantially upregulated in oral carcinoma, significantly associated with poor clinical outcomes and representing an independent prognostic predictor. Overexpression of LncHIFCAR induces pseudo-hypoxic gene signature, whereas knockdown of LncHIFCAR impairs the hypoxia-induced HIF-1α transactivation, sphere-forming ability, metabolic shift and metastatic potential in vitro and in vivo. Mechanistically, LncHIFCAR forms a complex with HIF-1α via direct binding and facilitates the recruitment of HIF-1α and p300 cofactor to the target promoters. Our results uncover an lncRNA-mediated mechanism for HIF-1 activation and establish the clinical values of LncHIFCAR in prognosis and potential therapeutic strategy for oral carcinoma.

7.
Mar Biotechnol (NY) ; 19(3): 310-319, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28500614

RESUMO

Virus infection often causes large amounts of mortality during teleost larvae stage. Strong induction of innate immunity to increase survival rates of teleost larvae has been less reported. In this study, we present a zebrafish IRF9-Stat2 fusion protein (zIRF9-S2C) as a strong innate immunity inducer and characterized induction of interferon-stimulated genes (ISGs) in zebrafish larvae. zIRF9-S2C could mimic IFN-stimulated gene factor 3 (ISGF3) complex to constitutively activate transcription of Mx promoter through IFN-stimulatory element (ISRE) sites. Mutation of two ISRE sites on Mx promoter reduced transactivation activities of Mx promoter induced by zIRF9-S2C. An electrophoretic mobility shift assay experiment shows that zIRF9-S2C could directly bind to two ISRE sites of Mx promoter. Induction of transactivation of Mx promoter by zIRF9-S2C shows significantly higher activity than by zebrafish IFN1 (zIFN1), IFNγ (zIFNγ), and Tetraodon IRF9-S2C (TnIRF9-S2C). zIRF9-S2C raises transcription of Mxa, Mxb, Mxc, Ifnφ1, Ifnφ2, and Ifnφ3 in zebrafish liver ((ZFL) cell line) cells and zebrafish larvae. Collectively, we suggest that IRF9-S2C could activate transcription of ISGs with species-specific recognition and could be an innate immunity inducer in teleost larvae.


Assuntos
Imunidade Inata , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Transcrição STAT2/genética , Peixe-Zebra/metabolismo , Animais , Células COS , Cercopithecus aethiops , Regulação da Expressão Gênica , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon/metabolismo , Larva/genética , Larva/imunologia , Larva/metabolismo , Proteínas de Resistência a Myxovirus/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Ativação Transcricional , Peixe-Zebra/genética , Peixe-Zebra/imunologia
8.
J Hematol Oncol ; 10(1): 33, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-28122633

RESUMO

BACKGROUND: Thrombospondin-2 (TSP-2) is a secreted matricellular glycoprotein that is found to mediate cell-to-extracellular matrix attachment and participates in many physiological and pathological processes. The expression profile of TSP-2 on tumors is controversial, and it up-regulates in some cancers, whereas it down-regulates in others, suggesting that the functional role of TSP-2 on tumors is still uncertain. METHODS: The expression of TSP-2 on prostate cancer progression was determined in the tissue array by the immunohistochemistry. The molecular mechanism of TSP-2 on prostate cancer (PCa) metastasis was investigated through pharmaceutical inhibitors, siRNAs, and miRNAs analyses. The role of TSP-2 on PCa metastasis in vivo was verified through xenograft in vivo imaging system. RESULTS: Based on the gene expression omnibus database and immunohistochemistry, we found that TSP-2 increased with the progression of PCa, especially in metastatic PCa and is correlated with the matrix metalloproteinase-2 (MMP-2) expression. Additionally, through binding to CD36 and integrin ανß3, TSP-2 increased cell migration and MMP-2 expression. With inhibition of p38, ERK, and JNK, the TSP-2-induced cell migration and MMP-2 expression were abolished, indicating that the TSP-2's effect on PCa is MAPK dependent. Moreover, the microRNA-376c (miR-376c) was significantly decreased by the TSP-2 treatment. Furthermore, the TSP-2-induced MMP-2 expression and the subsequent cell motility were suppressed upon miR-376c mimic stimulation. On the other hand, the animal studies revealed that the bone metastasis was abolished when TSP-2 was stably knocked down in PCa cells. CONCLUSIONS: Taken together, our results indicate that TSP-2 enhances the migration of PCa cells by increasing MMP-2 expression through down-regulation of miR-376c expression. Therefore, TSP-2 may represent a promising new target for treating PCa.


Assuntos
Neoplasias Ósseas/secundário , Metaloproteinase 2 da Matriz/genética , MicroRNAs/genética , Neoplasias da Próstata/patologia , Trombospondinas/fisiologia , Linhagem Celular , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Masculino
9.
Oncotarget ; 7(18): 26137-51, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27034167

RESUMO

Prostate cancer (PCa) with neuroendocrine differentiation (NED) is tightly associated with hormone refractory PCa (HRPC), an aggressive form of cancer that is nearly impossible to treat. Determining the mechanism of the development of NED may yield novel therapeutic strategies for HRPC. Here, we first demonstrate that repressor element-1 silencing transcription factor (REST), a transcriptional repressor of neuronal genes that has been implicated in androgen-deprivation and IL-6 induced NED, is essential for hypoxia-induced NED of PCa cells. Bioinformatics analysis of transcriptome profiles of REST knockdown during hypoxia treatment demonstrated that REST is a master regulator of hypoxia-induced genes. Gene set enrichment analysis (GSEA) of hypoxia and REST knockdown co-upregulated genes revealed their correlation with HRPC. Consistently, gene ontology (GO) analysis showed that REST reduction potential associated with hypoxia-induced tumorigenesis, NE development, and AMPK pathway activation. Emerging reports have revealed that AMPK activation is a potential mechanism for hypoxia-induced autophagy. In line with this, we demonstrate that REST knockdown alone is capable of activating AMPK and autophagy activation is essential for hypoxia-induced NED of PCa cells. Here, making using of in vitro cell-based assay for NED, we reveal a new role for the transcriptional repressor REST in hypoxia-induced NED and characterized a sequential molecular mechanism downstream of REST resulting in AMPK phosphorylation and autophagy activation, which may be a common signaling pathway leading to NED of PCa.


Assuntos
Autofagia , Diferenciação Celular , Transformação Celular Neoplásica/patologia , Hipóxia/fisiopatologia , Células Neuroendócrinas/patologia , Neoplasias da Próstata/patologia , Proteínas Repressoras/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Adesão Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Células Neuroendócrinas/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
10.
Fish Shellfish Immunol ; 38(1): 230-43, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24680831

RESUMO

Induction of interferons (IFNs) produces an innate immune response through activation of the JAK-STAT signaling pathway. Type I IFN signaling activates downstream gene expression through the IFN-stimulated gene factor 3 (ISGF3) complex, while type II IFN (IFN-γ) signaling is mediated through active STAT1 protein. The IFN target gene Mx is involved in the defense against viral infection. However, the mechanism by which Tetraodon (pufferfish) Mx is regulated by IFN signaling has not been identified. In this study, we describe the cloning and expression of Tetraodon STAT1, STAT2, and IFN regulatory factor 9 (IRF9). By combining constitutively-active STAT1 (STAT1-JH1) and STAT2 (STA2-JH1) fusion proteins with IRF9, we demonstrate that a constitutively-active ISGF3 complex increases the transcriptional activity of the Tetraodon Mx promoter via direct binding to two IFN-stimulated response element (ISRE) sites. In addition, a constitutively-active TnIRF9-S2C containing a fusion of the C-terminal region of STAT2 and IRF9 also activated the Mx promoter through binding to the ISRE sites. Furthermore, constitutively-active STAT1-JH1 elevates Mx promoter activity through two IFN gamma-activated sequence (GAS) elements. The Mx promoter is also activated by constitutively-active TnIRF9-S2C and STAT1-JH1 protein, as determined using an in vivo luciferase assay. We conclude that the Tetraodon Mx gene is activated via Type I (IFN-1) and Type II (IFN-γ) signaling. These results provide mechanistic insights into the role of IFN signaling in teleosts, and the in vivo luciferase assay may be suitable as a tool for studying induction and regulation by IFNs in teleost fish.


Assuntos
Regulação da Expressão Gênica/fisiologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Tetraodontiformes/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Cercopithecus aethiops , Clonagem Molecular , DNA Complementar , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Dados de Sequência Molecular , Proteínas de Resistência a Myxovirus/genética , Filogenia , Regiões Promotoras Genéticas , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genética , Transdução de Sinais
11.
Sci Signal ; 7(319): ra31, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24692592

RESUMO

Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer biosamples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.


Assuntos
Arginina/metabolismo , Argininossuccinato Sintase/deficiência , Neoplasias da Mama/metabolismo , Mitocôndrias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Arginina/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Hidrolases/farmacologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Polietilenoglicóis/farmacologia , Prognóstico , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS One ; 9(2): e88556, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551118

RESUMO

Prostate cancer (PCa) cells undergoing neuroendocrine differentiation (NED) are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE) tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ) markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA), a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results reveal the potential of targeting autophagy as part of a combined therapeutic regime for NE tumors.


Assuntos
Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interleucina-6/farmacologia , Células Neuroendócrinas/patologia , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Adenilato Quinase/metabolismo , Androgênios/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Proteína Beclina-1 , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cloroquina/farmacologia , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas de Membrana/metabolismo , Modelos Biológicos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células Neuroendócrinas/efeitos dos fármacos , Células Neuroendócrinas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/metabolismo
13.
PLoS One ; 9(1): e86345, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24466042

RESUMO

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Sistema da Linha Lateral/fisiologia , Morfogênese/genética , Proteínas da Mielina/genética , Receptores de Superfície Celular/genética , Via de Sinalização Wnt/genética , Proteínas de Peixe-Zebra/genética , Animais , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Nogo , Transdução de Sinais/genética , Peixe-Zebra
14.
Genes Cancer ; 1(1): 40-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20811583

RESUMO

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. The authors' laboratory has been interested in the role of the Src complex in prostate cancer progression, including the induction of androgen independence and metastasis. Previously, the authors reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells but rarely apoptosis. Here, they have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of the PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, the authors showed that a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth compared with the control group. Taken together, these data suggest that (1) autophagy serves a protective role in SFK inhibitor-mediated cell killing, and (2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors.

15.
Nucleic Acids Res ; 38(14): 4635-50, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20378713

RESUMO

In mammals, the Nogo family consists of Nogo-A, Nogo-B and Nogo-C. However, there are three Rtn-4/Nogo-related transcripts were identified in zebrafish. In addition to the common C-terminal region, the N-terminal regions of Rtn4-n/Nogo-C1, Rtn4-m/Nogo-C2 and Rtn4-l/Nogo-B, respectively, contain 9, 25 and 132 amino acid residues. In this study, we isolated the 5'-upstream region of each gene from a BAC clone and demonstrated that the putative promoter regions, P1-P3, are functional in cultured cells and zebrafish embryos. A transgenic zebrafish Tg(Nogo-B:GFP) line was generated using P1 promoter region to drive green fluorescent protein (GFP) expression through Tol2-mediated transgenesis. This line recapitulates the endogenous expression pattern of Rtn4-l/Nogo-B mRNA in the brain, brachial arches, eyes, muscle, liver and intestines. In contrast, GFP expressions by P2 and P3 promoters were localized to skeletal muscles of zebrafish embryos. Several GATA and E-box motifs are found in these promoter regions. Using morpholino knockdown experiments, GATA4 and GATA6 were involved in the control of P1 promoter activity in the liver and intestine, while Myf5 and MyoD for the control of P1 and P3 promoter activities in muscles. These data demonstrate that zebrafish Rtn4/Nogo transcripts might be generated by coupling mechanisms of alternative first exons and alternative promoter usage.


Assuntos
Proteínas da Mielina/genética , Regiões Promotoras Genéticas , Proteínas de Xenopus/genética , Peixe-Zebra/genética , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Embrião não Mamífero/metabolismo , Fatores de Transcrição GATA/metabolismo , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Camundongos , Proteínas da Mielina/metabolismo , Proteínas Nogo , Proteínas de Xenopus/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
16.
Fish Shellfish Immunol ; 28(5-6): 819-28, 2010 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20156562

RESUMO

In this paper, we report the cloning and characterization of the STAT6 gene from the pufferfish, Tetraodon nigroviridis. The TnSTAT6 gene is composed of 20 exons and 19 introns. The exon-intron organization of this gene is similar to that of HsSTAT6 except for the exons encoding the C-terminal transactivation domain. The full-length complementary (c)DNA of TnSTAT6 encodes a 794-amino acid protein that is 31% identical to human STAT6. We generated a constitutively active TnSTAT6-JH1 by fusing the kinase domain of carp JAK1 to the C-terminal end of TnSTAT6 and demonstrated that the fusion protein has specific DNA-binding ability and can activate a reporter construct carrying multiple copies of mammalian IL-4-response elements. Interestingly, TnSTAT6-JH1 associated with and phosphorylated TnSTAT6 on Tyr661. Mutation of this residue, Y661W, in TnSTAT6 abolished its association with TnSTAT6-JH1. This is consistent with the importance of the corresponding Tyr641 of HsSTAT6 in tyrosine phosphorylation and dimer formation. On the other hand, treatment of mammalian IL-4 did not induce tyrosine phosphorylation of wild-type TnSTAT6, suggesting that both the divergent N-terminal domain and coiled-coiled domain of TnSTAT6 may affect the interaction of TnSTAT6 with mammalian IL-4 receptor complexes.


Assuntos
Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Tetraodontiformes/genética , Tetraodontiformes/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Cercopithecus aethiops , Clonagem Molecular , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Interleucina-4/farmacologia , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Tirosina/metabolismo
17.
Fish Shellfish Immunol ; 28(5-6): 774-82, 2010 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20156563

RESUMO

In this study, we isolated and characterized both JAK and STAT genes from Artemia, Artemia franciscana. Although AfJAK showed only 19% identity (33% similarity) to the Drosophila Hop protein, AfJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7. On the other hand, AfSTAT showed higher identity (30%) to Drosophila STAT (STAT92E). The low identities of AfJAK and AfSTAT to Drosophila Hop and STAT92E suggest that JAK and STAT proteins are unique in each different species of invertebrate. RT-PCR analysis showed that both AfJAK and AfSTAT transcripts were ubiquitously expressed in the embryo, which is similar to the expression patterns of Drosophila Hop and STAT92E mRNAs during development. In addition, we generated a constitutively active form of AfSTAT by fusing the JH1 domain of AfJAK to the C-terminal end of AfSTAT. This fusion protein, AfSTAT-HA-JH1, autophosphorylated on its tyrosine residue and was able to bind to specific DNA motifs including the STAT-binding motifs in the Drosophila Raf promoter. Both AfJAK and AfSTAT proteins elicited the transactivation potential toward the fly Raf promoter in Sf9 cells. However, tyrosine phosphorylation of AfSTAT was not detected, which is consistent with the cellular localization analysis that most AfSTAT proteins were in the cytoplasm. Our results demonstrate that both JAK and STAT are present in the genome of Artemia, which can serve as the basis for further investigations to explore the role of the JAK/STAT signal pathway in the development and immune response of brine shrimp.


Assuntos
Artemia/genética , Artemia/metabolismo , Regulação da Expressão Gênica , Janus Quinases/genética , Janus Quinases/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Sequência de Aminoácidos , Animais , Artemia/citologia , Artemia/enzimologia , Células COS , Linhagem Celular , Cercopithecus aethiops , Clonagem Molecular , DNA/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Alinhamento de Sequência
18.
Development ; 135(5): 941-52, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18234726

RESUMO

We identified a zebrafish caudal-related homeobox (cdx1b) gene, which shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs.


Assuntos
Endoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Membrana/fisiologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Vetores Genéticos , Hibridização In Situ , Filogenia , Transdução de Sinais , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
19.
Dev Comp Immunol ; 32(7): 814-24, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18187191

RESUMO

In invertebrates, the JAK-STAT signaling pathway is involved in the anti-bacterial response and is part of an anti-viral response in Drosophila. In this study, we show that two STAT transcripts are generated by alternative splicing and encode two isoforms of Sf-STAT with different C-terminal ends. These two isoforms were produced and purified using the recombinant baculovirus technology. Both purified isoforms showed similar DNA-binding activity and displayed weak but significant transactivation potential toward a Drosophila promoter that contained a STAT-binding motif. No significant activation of the Sf-STAT protein in Sf9 cells was found by infection with baculovirus AcMNPV.


Assuntos
Expressão Gênica , Fatores de Transcrição STAT/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Fatores de Transcrição STAT/química , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Spodoptera , Ativação Transcricional/genética , Quinases raf/genética , Quinases raf/metabolismo
20.
FEBS Lett ; 581(22): 4265-71, 2007 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-17706649

RESUMO

In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as alphaA1- and betaA1-globin, but not the embryonic betae1-globin, was affected.


Assuntos
Eritropoetina/genética , Eritropoetina/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Cercopithecus aethiops , Clonagem Molecular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Células Eritroides/metabolismo , Eritropoetina/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hemoglobinas/biossíntese , Dados de Sequência Molecular , Especificidade de Órgãos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/química
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