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1.
Biol Res ; 53(1): 13, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293552

RESUMO

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.


Assuntos
Antígenos Glicosídicos Associados a Tumores/genética , Neoplasias da Vesícula Biliar/genética , Índios Sul-Americanos/genética , Animais , Antineoplásicos/farmacologia , Líquido Ascítico/metabolismo , Carcinogênese/genética , Testes de Carcinogenicidade , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Chile , Cisplatino/farmacologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Impressões Digitais de DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Células Epiteliais/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Perfilação da Expressão Gênica , Genes erbB-2/genética , Humanos , Queratina-19/genética , Queratina-7/genética , Masculino , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Análise de Sequência de RNA , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
2.
Cancer Sci ; 111(5): 1514-1527, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32030850

RESUMO

Estrogen-related receptor-α (ERRα) is a nuclear receptor of transcription factor that binds to estrogen responsive elements and estrogen-related responsive elements. Estrogen-related receptor-α is involved in metabolic processes and implicated in the progression and growth of several human malignancies. However, the biologic role and clinical significance of ERRα in gallbladder cancer (GBC) remains to be clarified. Here, we reported that ERRα protein expression was notably higher in GBC tissues than in cholecystitis tissues, and that the aberrantly higher ERRα expression was positively correlated with advanced TNM stage and indicated dismal prognosis of GBC (P < .01). In GBC cell lines NOZ and OCUG, the targeted depletion of ERRα retarded the growth and suppressed the migration and invasive capabilities of GBC cells, and inhibited epithelial-mesenchymal transition by decreasing the expression of mesenchymal markers and elevating the expression of epithelial markers. Moreover, ERRα knockdown inhibited tumor growth in nude mice and led to decreased expression levels of Nectin-4, p-PI3K p85α, and p-AKT. Overexpression of ERRα in the GBC-SD cell line showed exactly the opposite effect. The targeted inhibition of Nectin-4 antagonized GBC cell proliferation and invasion, which were induced by ERRα upregulation. Moreover, Nectin-4 depletion inhibited the ERRα-induced activation of the PI3K/AKT pathway. Chromatin immunoprecipitation analysis and dual-luciferase reporter gene assays showed that ERRα enhanced the transcription of Nectin-4 by binding to the promoter of Nectin-4. In conclusion, our data indicated that ERRα could be a potential target for the genetic treatment of GBC.


Assuntos
Moléculas de Adesão Celular/genética , Neoplasias da Vesícula Biliar/patologia , Receptores Estrogênicos/metabolismo , Transcrição Genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/genética , Feminino , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/mortalidade , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Estrogênicos/genética , Transdução de Sinais
3.
Cancer Sci ; 111(3): 817-825, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31925976

RESUMO

Recent studies have reported that tumor-infiltrating mast cells (TIM) play an important role in tumor regression, but the effect of TIM in gallbladder cancer (GBC) remains unclear. The present study aims to investigate the prognostic value of TIM in GBC patients and its responsiveness to gemcitabine-based adjuvant chemotherapy (ACT). A total of 298 GBC patients from Zhongshan Hospital were recruited for this study. TIM infiltration was measured by immunohistochemical staining. Accumulation of TIM is significantly associated with prolonged overall survival in GBC patients. The benefit from gemcitabine-based ACT was superior among patients with high infiltration of TIM with GBC. Multivariate analysis identified TIM infiltration as an independent prognostic factor for overall survival. A heatmap showed that TIM-activated gene signatures were positively correlated with CD8+ T cells' gene signatures. Gene set enrichment analysis (GSEA) suggested that TIM was related to multiple T cell-related processes and signaling pathways, including the interferon gamma signaling pathway and the leukocyte migration signaling pathway. It was confirmed that CD8+ T cell infiltration was positively correlated with high TIM infiltration in tissue microarray (TMA), suggesting that TIM infiltration was linked to the immune surveillance in GBC. TIM can be used as an independent prognostic factor and a predictor of therapeutic response of gemcitabine-based ACT in GBC patients, which may mediate immune surveillance by recruiting and activating CD8+ T cells in GBC.


Assuntos
Neoplasias da Vesícula Biliar/patologia , Linfócitos do Interstício Tumoral/patologia , Mastócitos/patologia , Antimetabólitos Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Quimioterapia Adjuvante/métodos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Humanos , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais/efeitos dos fármacos
4.
Nat Commun ; 10(1): 5492, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792210

RESUMO

Gemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. Here, we utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNA-modifying enzymes. Mechanistically, loss of ELP5 impairs the integrity and stability of the Elongator complex to abrogate wobble U34 tRNA modification, and directly impedes the wobble U34 modification-dependent translation of hnRNPQ mRNA, a validated P53 internal ribosomal entry site (IRES) trans-acting factor. Downregulated hnRNPQ is unable to drive P53 IRES-dependent translation, but rescuing a U34 modification-independent hnRNPQ mutant could restore P53 translation and gemcitabine sensitivity in ELP5-depleted GBC cells. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy. These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells.


Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Proteínas de Transporte/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxicitidina/análogos & derivados , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Estudos de Coortes , Desoxicitidina/administração & dosagem , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Estudo de Associação Genômica Ampla , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal , Masculino , Pessoa de Meia-Idade , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Mol Cancer ; 18(1): 166, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752867

RESUMO

BACKGROUND: CircRNAs are found to affect initiation and progression of several cancer types. However, whether circRNAs are implicated in gallbladder cancer (GBC) progression remains obscure. METHODS: We perform RNA sequencing in 10 pairs of GBC and para-cancer tissues. CCK8 and clone formation assays are used to evaluate proliferation ability of GBC cells. qPCR and Western blot are used to determine expression of RNAs and proteins, respectively. CircRNA-protein interaction is confirmed by RNA pulldown, RNA immunoprecipitation, and fluorescence in situ hybridization. RESULTS: We find that circRNA expression pattern is tremendously changed during GBC development. Among dozens of significantly changed circRNAs, a circRNA generated from the oncogene ERBB2, named as circERBB2, is one of the most significant changes. CircERBB2 promotes GBC proliferation, in vitro and in vivo. Other than being a miRNA sponge, circERBB2 accumulates in the nucleoli and regulates ribosomal DNA transcription, which is one of the rate-limiting steps of ribosome synthesis and cellular proliferation. CircERBB2 regulates nucleolar localization of PA2G4, thereby forming a circERBB2-PA2G4-TIFIA regulatory axis to modulate ribosomal DNA transcription and GBC proliferation. Increased expression of circERBB2 is associated with worse prognosis of GBC patients. CONCLUSIONS: Our findings demonstrate that circERBB2 serves as an important regulator of cancer cell proliferation and shows the potential to be a new therapeutic target of GBC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Ribossômico , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Regulação Neoplásica da Expressão Gênica , RNA Circular , Proteínas de Ligação a RNA/metabolismo , Receptor ErbB-2/genética , Processamento Alternativo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Progressão da Doença , Neoplasias da Vesícula Biliar/patologia , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Prognóstico , Curva ROC
6.
Mol Cancer ; 18(1): 167, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752906

RESUMO

BACKGROUNDS: Long non-coding RNAs (lncRNAs) are essential factors that regulate tumor development and metastasis via diverse molecular mechanisms in a broad type of cancers. However, the pathological roles of lncRNAs in gallbladder carcinoma (GBC) remain largely unknown. Here we discovered a novel lncRNA termed lncRNA Highly expressed in GBC (lncRNA-HGBC) which was upregulated in GBC tissue and aimed to investigate its role and regulatory mechanism in the development and progression of GBC. METHODS: The expression level of lncRNA-HGBC in GBC tissue and different cell lines was determined by quantitative real-time PCR. The full length of lncRNA-HGBC was obtained by 5' and 3' rapid amplification of the cDNA ends (RACE). Cellular localization of lncRNA-HGBC was detected by fluorescence in situ hybridization (FISH) assays and subcellular fractionation assay. In vitro and in vivo assays were preformed to explore the biological effects of lncRNA-HGBC in GBC cells. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation (RIP) assay were used to identify lncRNA-HGBC-interacting proteins. Dual luciferase reporter assays, AGO2-RIP, and MS2-RIP assays were performed to verify the interaction between lncRNA-HGBC and miR-502-3p. RESULTS: We found that lncRNA-HGBC was upregulated in GBC and its upregulation could predict poor survival. Overexpression or knockdown of lncRNA-HGBC in GBC cell lines resulted in increased or decreased, respectively, cell proliferation and invasion in vitro and in xenografted tumors. LncRNA-HGBC specifically bound to RNA binding protein Hu Antigen R (HuR) that in turn stabilized lncRNA-HGBC. LncRNA-HGBC functioned as a competitive endogenous RNA to bind to miR-502-3p that inhibits target gene SET. Overexpression, knockdown or mutation of lncRNA-HGBC altered the inhibitory effects of miR-502-3p on SET expression and downstream activation of AKT. Clinically, lncRNA-HGBC expression was negatively correlated with miR-502-3p, but positively correlated with SET and HuR in GBC tissue. CONCLUSIONS: Our study demonstrates that lncRNA-HGBC promotes GBC metastasis via activation of the miR-502-3p-SET-AKT cascade, pointing to lncRNA-HGBC as a new prognostic predictor and a therapeutic target.


Assuntos
Proteínas de Ligação a DNA/genética , Proteína Semelhante a ELAV 1/genética , Neoplasias da Vesícula Biliar/genética , Regulação Neoplásica da Expressão Gênica , Chaperonas de Histonas/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Chaperonas de Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA
7.
J Cancer Res Clin Oncol ; 145(11): 2699-2711, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31586263

RESUMO

BACKGROUND: Gallbladder cancer (GBC) is a rare neoplasia of the biliary tract with high mortality rates and poor prognosis. Signs and symptoms of GBC are not specific and often arise at late stage of disease. For this reason, diagnosis is typically made when the cancer is already in advanced stages, and prognosis for survival is less than 5 years in 90% of cases. Biomarkers to monitor disease progression and novel therapeutic alternative targets for these tumors are strongly required. Commonly, dysregulated protein synthesis contributes to carcinogenesis and cancer progression. In this case, protein synthesis directs translation of specific mRNAs, and, in turn, promotes cell survival, invasion, angiogenesis, and metastasis of tumors. In eukaryotes, protein synthesis is regulated at its initiation, which is a rate-limiting step involving eukaryotic translation initiation factors (eIFs). We hypothesize that eIFs represent crossroads in the development of GBC, and might serve as potential biomarkers. The study focus was the role of eIF6 (an anti-association factor for the ribosomal subunits) in GBC. METHODS: In human GBC samples, the expression of eIF6 was analyzed biochemically at the protein (immunohistochemistry, immunoblot analyses) and mRNA levels (qRT-PCR). RESULTS: High levels of eIF6 correlated with shorter overall survival in biliary tract cancer (BTC) patients (n = 28). Immunohistochemical data from tissue microarrays (n = 114) demonstrated significantly higher expression levels of eIF6 in GBC compared to non-neoplastic tissue. Higher eIF6 expression on protein (immunoblot) and mRNA (qRT-PCR) level was confirmed by analyzing fresh frozen GBC patient samples (n = 14). Depletion of eIF6 (using specific siRNA-mediated knockdown) in Mz-ChA-2 and TFK-1 cell lines inhibited cell proliferation and induced apoptosis. CONCLUSION: Our data indicates that eIF6 overexpression plays a major role in the translational control of GBC, and indicates its potential as a new biomarker and therapeutic target in GBC.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Neoplasias da Vesícula Biliar/patologia , Regulação Neoplásica da Expressão Gênica , Idoso , Apoptose , Proliferação de Células , Fatores de Iniciação em Eucariotos/genética , Feminino , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Humanos , Masculino , Prognóstico , Células Tumorais Cultivadas
8.
Mol Cancer ; 18(1): 145, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31623628

RESUMO

BACKGROUND: Circular RNAs (circRNAs) have recently been identified as potential functional modulators of the cellular physiology processes. The study aims to uncover the potential clinical value and driving molecular mechanisms of circRNAs in gallbladder cancer (GBC). PATIENTS AND METHODS: We performed RNA sequencing from four GBC and paired adjacent normal tissues to analyze the circRNA candidates. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to measure the circFOXP1 expression from 40 patient tissue samples. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro and in vivo assays were performed to prove the functional significance of circFOXP1. Double luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed. RESULTS: By performing RNA sequencing from GBC and paired adjacent normal tissues to analyze the circRNA candidates, we identified that circFOXP1 (hsa_circ_0008234) expression was significantly upregulated in GBC tissues and positively associated with lymph node metastasis, advanced TNM stage and poor prognosis in patients. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro assays demonstrated that circFOXP1 has pleiotropic effects, including promotion of cell proliferation, migration, invasion, and inhibition of cell apoptosis in GBC. In vivo, circFOXP1 promoted tumor growth. Mechanistically, double luciferase reporter, RNA immunoprecipitation (RIP) and biotin-labeled RNA pull-down assays clarified that circFOXP1 interacted with PTBP1 that could bind to the 3'UTR region and coding region (CDS) of enzyme pyruvate kinase, liver and RBC (PKLR) mRNA (UCUU binding bites) to protect PKLR mRNA from decay. Additionally, circFOXP1 acted as the sponge of miR-370 to regulate PKLR, resulting in promoting Warburg effect in GBC progression. CONCLUSIONS: These results demonstrated that circFOXP1 serve as a prognostic biomarker and critical regulator in GBC progression and Warburg effect, suggesting a potential target for GBC treatment.


Assuntos
Metabolismo Energético , Fatores de Transcrição Forkhead/genética , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Regulação Neoplásica da Expressão Gênica , Piruvato Quinase/genética , RNA Circular , Proteínas Repressoras/genética , Regiões 3' não Traduzidas , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Neoplasias da Vesícula Biliar/mortalidade , Neoplasias da Vesícula Biliar/patologia , Perfilação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Modelos de Riscos Proporcionais , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Surg Oncol ; 31: 83-89, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31541911

RESUMO

The most common malignancy of the biliary tract, gallbladder cancer (GBC) often has a dismal prognosis. The aggressive nature of the tumor, delayed diagnosis at advanced stages of the disease, and lack of effective treatment options are some of the factors that contribute to a poor outcome. Early detection and accurate assessment of disease burden is critical to optimize management and improve long-term survival, as well as identify patients for adjuvant therapy and clinical trials. With recent advances in the understanding of the molecular pathogenesis of GBC, several specific diagnostic and biomarkers have been proposed as being of diagnostic and prognostic importance. Indeed, identification of novel diagnostic and prognostic markers has an important role in early diagnosis and development of targeted therapies among patients with GBC. Next-generation sequencing technology and genomewide data analysis have provided novel insight into understanding the molecular pathogenesis of biliary tract cancers, thereby identifying potential biomarkers for clinical use. We herein review available GBC biomarkers and the potential clinical implications in the management of GBC.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Vesícula Biliar/diagnóstico , Neoplasias da Vesícula Biliar/terapia , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Animais , Neoplasias da Vesícula Biliar/metabolismo , Humanos , Prognóstico
10.
Cancer Sci ; 110(11): 3510-3519, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31487418

RESUMO

NOP2/Sun domain family, member 2 (NSUN2) is a nuclear RNA methyl-transferase catalyzing 5-methylcytosine formation. Evidence shows that NSUN2 is correlated with cell unlimited proliferation. However, its functional role in gallbladder carcinoma (GBC), which is the most common biliary tract malignancy and has a poor prognosis, remains to be determined. Here we found that NSUN2 was highly expressed in GBC tissues as well as cell lines. NSUN2 silencing repressed GBC cell proliferation and tumorigenesis both in vitro and in vivo. Conversely, upregulation of NSUN2 enhanced GBC cell growth and colony formation. We further discovered that RPL6 was a closely interacting partner with NSUN2. Silencing RPL6 resulted in insufficient NSUN2 translational level and accumulative NSUN2 transcriptional level. Exogenous expression of NSUN2 partially rescued the effect of RPL6 in gallbladder cancer progression. Taken together, our data provided novel mechanic insights into the function of NSUN2 in GBC, thus pointing to NSUN2 as a potential and effective therapeutic approach to GBC treatment.


Assuntos
Carcinoma/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Metiltransferases/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Carcinoma/patologia , Carcinoma/terapia , Linhagem Celular Tumoral , Proliferação de Células , Colecistite/metabolismo , Progressão da Doença , Neoplasias da Vesícula Biliar/patologia , Neoplasias da Vesícula Biliar/terapia , Humanos , Metiltransferases/antagonistas & inibidores , Camundongos , Camundongos Nus , Ensaio Tumoral de Célula-Tronco , Regulação para Cima
11.
Am J Physiol Gastrointest Liver Physiol ; 317(5): G670-G681, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31369289

RESUMO

In recent studies, microRNAs (miRs) have been widely explored as important regulators in tumor suppression. miR-136 has been suggested to participate in tumor inhibition through control of vital cellular processes, such as angiogenesis, proliferation, and apoptosis. This study aimed to evaluate the effects of overexpressed miR-136 by transferring mimics in gallbladder cancer (GBC) and to assess the functional role of miR-136 in GBC cell behaviors with the involvement of the mitogen-activated protein kinase kinase 4 (MAP2K4)-dependent JNK signaling pathway. Differentially expressed miRs associated with GBC were screened using microarray expression profiles, which identified that miR-136 expression was decreased in GBC. Furthermore, MAP2K4 was validated as a target gene of miR-136. To uncover functional relevance regarding miR-136 and MAP2K4 in GBC, cultured GBC cell lines were prepared to transfect with mimic, inhibitor, siRNA, or vectors. At the same time, the transfected GBC cells were inoculated into nude mice to validate findings in vivo. The obtained results demonstrated that overexpressed miR-136 inhibited angiogenesis and cell proliferation and promoted apoptosis in GBC cell lines in vitro, accompanied by impeded cellular tumorigenicity in nude mice via the suppression of MAP2K4. Moreover, the overexpression of MAP2K4 and the activation of the JNK signaling pathway reversed the inhibitory effects of miR-136 on the angiogenesis and tumorigenicity of GBC cells. Together, our results indicated that overexpressed miR-136 attenuates angiogenesis and enhances cell apoptosis in GBC via the JNK signaling pathway by downregulating the expression of MAP2K4.NEW & NOTEWORTHY This study is based on previous studies suggesting the tumor-suppressive role of microRNA (miR)-136 in various cancers. We aim to clarify whether miR-136 could function as a tumor suppressor in gallbladder cancer (GBC) and an underlying mechanism. In vitro and in vivo assays delineated that the tumor-suppressive role of miR-136 in GBC is achieved through inactivation of the JNK signaling pathway by downregulation of MAP2K4.


Assuntos
Neoplasias da Vesícula Biliar/genética , Sistema de Sinalização das MAP Quinases , MicroRNAs/genética , Animais , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Humanos , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Regulação para Cima
12.
Methods Mol Biol ; 2037: 231-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31463849

RESUMO

Gallbladder cancer (GBC) is a common form of malignancy, which has high incidence rates in the northern parts of India, South America, Japan, and in Native American populations. A few metabolomic studies have revealed changes in the composition of biofluids, tissues, and gallstones by nuclear magnetic resonance (NMR), which may prove useful in understanding GBC. In this chapter, we focus on the use of NMR in unveiling the metabolomics of GBC.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Metabolômica/métodos , Biomarcadores Tumorais/análise , Humanos
13.
Biosci Biotechnol Biochem ; 83(12): 2257-2264, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31411121

RESUMO

Gallbladder carcinoma (GBC) is a highly lethal malignancy of the gastrointestinal tract. Despite extensive research, the underlying molecular mechanism of GBC remains largely unclear. Deleted in malignant brain tumors 1 (DMBT1) is low-expression during cancer progression and as a potential tumor-suppressor gene in various types of cancer. However, its role in Gallbladder cancer remains poorly understood. Here, we found that DMBT1 was significantly low-expression and deletion of copy number in GBC tissues by qRT-PCR and Western blot. Overexpression of DMBT1 impaired survival, promoted apoptosis in GBC cells in vitro, and inhibited tumor progression in vivo. Further study of underlying mechanisms demonstrated that DMBT1 combined with PTEN which could stabilize PTEN protein, resulting in inhibiting the activation of PI3K/AKT signaling pathway. Our study revealed a new sight of DMBT1 as a tumor-suppressor gene on the PI3K/AKT pathway in GBC, which may be a potential therapeutic target for improving treatment.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Vesícula Biliar/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Neoplasias da Vesícula Biliar/enzimologia , Neoplasias da Vesícula Biliar/metabolismo , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Supressoras de Tumor/genética
14.
Yonsei Med J ; 60(7): 604-610, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31250573

RESUMO

PURPOSE: This study aimed to determine the prognostic value of new quantitative parameters of ¹8F-fluorodeoxyglucose positron emission tomography/computed tomography (¹8F-FDG PET/CT), including metabolic tumor volume (MTV), in patients with locally advanced and metastatic gallbladder cancer (GBC). MATERIALS AND METHODS: In total, 83 patients initially diagnosed with locally advanced and metastatic GBC and who underwent ¹8F-FDG PET/CT at the time of initial diagnosis were retrospectively reviewed. The metabolic volume-based PET parameters of primary tumors and metastatic lesions were measured, including maximum and average standardized uptake values (SUV), MTV, and total lesion glycolysis. An overall survival (OS) analysis was performed using the Kaplan-Meier method with PET and clinical parameters. A Cox proportional hazards regression analysis was performed to determine independent prognostic factors. RESULTS: In univariate analysis, pathologic differentiation (p<0.001), performance status (PS; p=0.003), C-reactive protein (CRP) level (p=0.009), and PET-related SUVmt max (the highest SUV among the metastatic lesions) (p=0.040) and MTVtotal (the sum of the MTVs of both the primary and metastatic lesions) (p=0.031), were significant for OS. In multivariate analysis, MTVtotal (hazard ratio: 2.07; 95% confidence interval: 1.23-3.48; p=0.006) remained significant for the prediction of OS, as did differentiation (p=0.001), PS (p=0.001), and CRP (p=0.039). CONCLUSION: In locally advanced and metastatic GBC, volume-based PET/CT parameters of the total tumor burden of malignancy, such as MTVtotal, were found to be useful for the identification of patients with poor prognosis.


Assuntos
Fluordesoxiglucose F18 , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Glicólise , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Carga Tumoral , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Neoplasias da Vesícula Biliar/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos
15.
J Exp Clin Cancer Res ; 38(1): 247, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182136

RESUMO

BACKGROUND: Gallbladder cancer (GBC) is an extremely malignant tumor with a high mortality rate. Little is known about its invasion and metastasis mechanism so far. METHODS: To identify the driver genes in GBC metastasis, we performed a mRNA microarray of metastatic GBC and paired non-tumor samples, and found PLEK2 was markedly upregulated in GBC tissues. Next, the expression of PLEK2 in GBC were examined in a larger cohort of patients by qRT-PCR, western blot and IHC staining. The clinicopathologic correlation of PLEK2 was determined by statistical analyses. The biological involvement of PLEK2 in GBC metastasis and the underlying mechanisms were investigated. RESULTS: In this study, we found that PLEK2 had higher expression in GBC tumor tissues compared to non-cancerous adjacent tissues and cholecystolithiasis tissues. The clinicopathologic analyses showed PLEK2 expression was positively correlated with tumor TNM stage, distant metastasis and PLEK2 was an independent predictor of overall survival (OS) in GBC patients. The cellular function assays showed PLEK2 promoted GBC cells migration, invasion and liver metastasis in mouse model via the regulation of epithelial-mesenchymal transition (EMT) process. Our mass spectrum and co-immunoprecipitation (co-IP) assays demonstrated that PLEK2 could interact with the kinase domain of EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, RNA-sequencing and qRT-PCR results demonstrated chemokine (C-C motif) ligand 2 (CCL2), a target gene downstream of PLEK2/EGFR signaling, mediated the motility-promoting function of PLEK2. CONCLUSIONS: On the basis of these collective data, we propose that PLEK2 promotes the invasion and metastasis of GBC by EGFR/CCL2 pathway and PLEK2 can serve as a potential therapeutic target for GBC treatment.


Assuntos
Quimiocina CCL2/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Proteínas de Membrana/metabolismo , Transdução de Sinais , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Receptores ErbB/metabolismo , Neoplasias da Vesícula Biliar/mortalidade , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Ligação Proteica
16.
J Exp Clin Cancer Res ; 38(1): 244, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174563

RESUMO

BACKGROUND: Gallbladder cancer (GBC) is the most malignant cancer occurring in the biliary tract cancer featured with undesirable prognosis, in which most patients die within a year of cholecystectomy. Long noncoding RNAs (lncRNAs) function as critical regulators of multiple stages of cancers. Herein, the mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in GBC is investigated. METHODS: Microarray-based analysis initially provided data suggesting that the expression of MALAT1 was up-regulated while that of the ABI family member 3 binding protein (ABI3BP) was down-regulated in GBC tissues and cell lines. Kaplan-Meier method was then adopted to analyze the relationship between the MALAT1 expression and overall survival and disease-free survival of patients with GBC. A set of in vitro and in vivo experiments were conducted by transducing ABI3BP-vector or sh-MALAT1 into GBC cells. RESULTS: The results confirmed that the cancer prevention effects triggered by restored ABI3BP and depleted MALAT1 as evidenced by suppressed cell growth and enhanced cell senescence. MALAT1 was observed to down-regulate ABI3BP expression through recruitment of the enhancer of zeste homolog 2 (EZH2) to the ABI3BP promoter region while the silencing of MALAT1 or suppression of H3K27 methylation was observed to promote the expression of ABI3BP. Furthermore, GBC patients with high expression of MALAT1 indicated poor prognosis. CONCLUSION: The current study clarifies that MALAT1 silencing and ABI3BP elevation impede the GBC development through the H3K27 methylation suppression induced by EZH2, highlighting a promising competitive paradigm for therapeutic approaches of GBC.


Assuntos
Proteínas de Transporte/genética , Senescência Celular/genética , Neoplasias da Vesícula Biliar/genética , Regulação Neoplásica da Expressão Gênica , Interferência de RNA , RNA Longo não Codificante/genética , Adulto , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Xenoenxertos , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Prognóstico , Adulto Jovem
17.
Am J Clin Pathol ; 152(2): 190-199, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31107526

RESUMO

OBJECTIVES: Albumin messenger RNA (mRNA) expression is a marker of hepatocellular differentiation. Most published data are from review of tissue microarrays, and albumin in situ hybridization (ISH) expression across several tumor types is incompletely characterized. METHODS: Sections from 221 tumors were evaluated for albumin mRNA. Immunohistochemistry was used to confirm diagnoses. Albumin ISH was performed according to manufacturer-provided instructions. Fifty-nine cases were evaluated with both commercial ISH assays. RESULTS: Albumin mRNA was detected in all hepatocellular carcinomas (HCCs) and 81% of intrahepatic cholangiocarcinomas. Lung (20%), gallbladder (39%), hepatoid pancreatic (n = 1 of 1) adenocarcinoma, breast invasive ductal carcinoma (18%), yolk sac tumor (25%), and acinar cell carcinoma (29%) showed expression. Both assays were concordant in 93% of cases. CONCLUSIONS: Albumin ISH was expressed in all HCCs studied. It was also positive in intrahepatic cholangiocarcinoma and patchy positive in gallbladder adenocarcinoma and a subset of other neoplasms, which can be a potential pitfall.


Assuntos
Adenocarcinoma/metabolismo , Albuminas/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Carcinoma Hepatocelular/metabolismo , Colangiocarcinoma/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias Hepáticas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Albuminas/genética , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/patologia , Humanos , Hibridização In Situ , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Estudos Retrospectivos
18.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 607-614, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31074773

RESUMO

Gallbladder carcinoma (GBC) is the most common and aggressive cancer of the biliary tract. Liensinine has been proved to have hypotensive effect. However, the effect of liensinine on GBC is still unknown. The aim of this study is to investigate the effect and mechanism of liensinine in human GBC cells. Cell viability assay and colony formation assay were performed to assess cell growth and proliferation. Flow cytometry analysis was used to investigate cell cycle apoptosis in vitro. Hoechst 33342 staining was also used to evaluate cell apoptosis. Western blot analysis was used to determine the expression of proteins corresponding to the related cell cycle and apoptosis. The effect of liensinine treatment in vivo was experimented with xenografted tumors. We found that liensinine significantly inhibited the growth of GBC cells both in vivo and in vitro. In vitro, cell growth and proliferation were significantly suppressed by liensinine in a dose- and time-dependent manner. In vivo, liensinine inhibited tumor growth. Liensinine could induce GBC cells G2/M phase arrest by up-regulating the levels of Cyclin B1 and CDK1 proteins. Liensinine also affected GBC cell cycle progression and induced apoptosis by down-regulating phosphorylated protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), phosphatidylinositol 3-kinase (PI3K), and Zinc finger X-chromosomal protein (ZFX) proteins. Liensinine induced G2/M arrest and apoptosis in gallbladder cancer, suggesting that liensinine might represent a novel and effective agent against gallbladder cancer.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Neoplasias da Vesícula Biliar/tratamento farmacológico , Isoquinolinas/farmacologia , Fenóis/farmacologia , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Humanos , Isoquinolinas/química , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Nelumbo/química , Fenóis/química , Fosfatidilinositol 3-Quinases/metabolismo , Fitoterapia , Proteínas Proto-Oncogênicas c-akt/metabolismo
19.
Mol Cancer ; 18(1): 82, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953511

RESUMO

BACKGROUND: Gallbladder cancer is the most common biliary tract malignancy and not sensitive to chemotherapy. Autophagy is an important factor prolonging the survival of cancer cells under chemotherapeutic stress. We aimed to investigate the role of long non-coding RNAs (lncRNAs) in autophagy and chemoresistance of gallbladder cancer cells. METHODS: We established doxorubicin (Dox)-resistant gallbladder cancer cells and used microarray analysis to compare the expression profiles of lncRNAs in Dox-resistant gallbladder cancer cells and their parental cells. Knockdown or exogenous expression of lncRNA combined with in vitro and in vivo assays were performed to prove the functional significance of lncRNA. The effects of lncRNA on autophagy were assessed by stubRFP-sensGFP-LC3 and western blot. We used RNA pull-down and mass spectrometry analysis to identify the target proteins of lncRNA. RESULTS: The drug-resistant property of gallbladder cancer cells is related to their enhanced autophagic activity. And we found a lncRNA ENST00000425894 termed gallbladder cancer drug resistance-associated lncRNA1 (GBCDRlnc1) that serves as a critical regulator in gallbladder cancer chemoresistance. Furthermore, we discovered that GBCDRlnc1 is upregulated in gallbladder cancer tissues. Knockdown of GBCDRlnc1, via inhibiting autophagy at initial stage, enhanced the sensitivity of Dox-resistant gallbladder cancer cells to Dox in vitro and in vivo. Mechanically, we identified that GBCDRlnc1 interacts with phosphoglycerate kinase 1 and inhibits its ubiquitination in Dox-resistant gallbladder cancer cells, which leads to the down-regulation of autophagy initiator ATG5-ATG12 conjugate. CONCLUSIONS: Our findings established that the chemoresistant driver GBCDRlnc1 might be a candidate therapeutic target for the treatment of advanced gallbladder cancer.


Assuntos
Autofagia/genética , Neoplasias da Vesícula Biliar/genética , Regulação Neoplásica da Expressão Gênica , Fosfoglicerato Quinase/genética , RNA Longo não Codificante/genética , Idoso , Animais , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Fosfoglicerato Quinase/metabolismo , RNA Longo não Codificante/agonistas , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Biomed Res Int ; 2019: 9205851, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019975

RESUMO

The effects of standard clinical therapies including surgery and chemotherapy are poor in advanced gallbladder cancer (GBC). There are a few reported cases of human epidermal growth factor receptor 2 (HER2)-positive GBC that responded well to trastuzumab. But trastuzumab has not yet been used to treat HER2-negative GBC. In this study, we investigated the cytotoxic effects of different combined therapies with trastuzumab and gemcitabine and/or 5-fluorouracil on HER2-negative GBC cell lines in vitro and in vivo. Trastuzumab alone showed almost no cytotoxicity to GBC cells with originally low HER2 gene amplification. Sequential therapy with chemotherapy followed by trastuzumab showed superiority over reverse sequential chemotherapy (P<0.05), concurrent combined chemotherapy (P<0.05), chemotherapy alone (P<0.05), and trastuzumab alone (P<0.05) in terms of cytotoxicity. Sequential therapy with chemotherapy followed by trastuzumab nearly completely inhibited cell viability in HER2-negative GBC cells. Similar results were observed with regard to apoptosis. Western blot analysis showed that gemcitabine/5-fluorouracil increased the expressions of total and phosphorylated forms of HER2, thus enhancing the cytotoxicity of trastuzumab. In vivo study verified the results of in vitro study by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis. Moreover, not only the lightest tumor bearing but also the best survival state was detected in sequential therapy with chemotherapy followed by trastuzumab group compared with other groups. Our in vivo and in vitro data suggest that sequential therapy with gemcitabine/5-fluorouracil followed by trastuzumab represents a novel and promising therapeutic strategy against HER2-negative GBC. The upregulation of phosphorylated HER2 and phosphorylated-AKT induced by gemcitabine/5-fluorouracil treatment shows that HER2/AKT pathway is triggered.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Vesícula Biliar , Animais , Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Feminino , Fluoruracila/farmacologia , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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