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1.
Blood Cancer J ; 13(1): 23, 2023 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-36737429

RESUMO

Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a "proof of concept", we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.


Assuntos
Neoplasias Hematológicas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Processamento Alternativo , RNA , Splicing de RNA
3.
Pancreatology ; 22(2): 294-303, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35120820

RESUMO

BACKGROUND & AIMS: Sca-1 is a surface marker for murine hematopoietic stem cells (HSCs) and type-I interferon is a key regulator for Lin-Sca-1+ HSCs expansion through Ifnar/Stat-1/Sca-1-signaling. In this study we aimed to characterize the role and regulation of Sca-1+ cells in pancreatic regeneration. METHODS: To characterize Sca-1 in vivo, immunohistochemistry and immunofluorescence staining of Sca-1 was conducted in normal pancreas, in cerulein-mediated acute pancreatitis, and in Kras-triggered cancerous lesions. Ifnar/Stat-1/Sca-1-signaling was studied in type-I IFN-treated epithelial explants of adult wildtype, Ifnar-/-, and Stat-1-/- mice. Sca-1 induction was analyzed by gene expression and FACS analysis. After isolation of pancreatic epithelial Lin-Sca-1+cells, pancreatosphere-formation and immunofluorescence-assays were carried out to investigate self-renewal and differentiation capabilities. RESULTS: Sca-1+ cells were located in periacinar and periductal spaces and showed an enrichment during cerulein-induced acute pancreatitis (23.2/100 µm2 ± 4.9 SEM) and in early inflammation-mediated carcinogenic lesions of the pancreas of KrasG12D mice (35.8/100 µm2 ± SEM 1.9) compared to controls (3.6/100 µm2 ± 1.3 SEM). Pancreatic Lin-Sca-1+ cells displayed a small population of 1.46% ± 0.12 SEM in FACS. In IFN-ß treated pancreatic epithelial explants, Sca-1 expression was increased, and Lin-Sca-1+ cells were enriched in vitro (from 1.49% ± 0.36 SEM to 3.85% ± 0.78 SEM). Lin-Sca-1+ cells showed a 12 to 51-fold higher capacity for clonal self-renewal compared to Lin-Sca-1- cells and generated cells express markers of the acinar and ductal compartment. CONCLUSIONS: Pancreatic Sca-1+ cells enriched during parenchymal damage showed a significant capacity for cell renewal and in vitro plasticity, suggesting that corresponding to the type I interferon-dependent regulation of Lin-Sca-1+ hematopoietic stem cells, pancreatic Sca-1+ cells also employ type-I-interferon for regulating progenitor-cell-homeostasis.


Assuntos
Plasticidade Celular , Pancreatite , Doença Aguda , Animais , Antígenos Ly/análise , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células Epiteliais , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologia
4.
Leukemia ; 36(4): 1088-1101, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35082402

RESUMO

Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds are early events in MM pathogenesis, enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers, and here we tested anti-MM effects of the Erk1/2 + CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC < 0.5) cytotoxicity of Erk1/2i + CDK4/6i in MM-cells. Erk1/2i + CDK4/6i treatment in a dose-dependent manner arrested MM-cells in the G0/G1 phase and activated mitochondrial apoptotic signaling. Our studies showed that Erk1/2i + CDK4/6i treatment-induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling, such as c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of these inhibitors. We identified Erk1/2i + CDK4/6i treatment associated five-gene signature which includes SNRPB and SLC25A5; these genes are involved in RNA processing and mitochondrial metabolism, respectively. Overall, our studies provide the preclinical framework for Erk1/2i + CDK4/6i combination clinical trials to target Ras+CDK pathways to improve patient outcome in MM.


Assuntos
Neoplasias da Mama , Mieloma Múltiplo , Neoplasias da Mama/tratamento farmacológico , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Feminino , Humanos , Mieloma Múltiplo/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico
5.
Viruses ; 13(10)2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34696475

RESUMO

Since the beginning of the 20th century, bacteriophages (phages), i.e., viruses that infect bacteria, have been used as antimicrobial agents for treating various infections. Phage preparations targeting a number of bacterial pathogens are still in use in the post-Soviet states and are experiencing a revival in the Western world. However, phages have never been used to treat diseases caused by Bacteroides fragilis, the leading agent cultured in anaerobic abscesses and postoperative peritonitis. Enterotoxin-producing strains of B. fragilis have been associated with the development of inflammatory diarrhea and colorectal carcinoma. In this study, we evaluated the molecular biosafety and antimicrobial properties of novel phage species vB_BfrS_VA7 (VA7) lysate, as well as its impact on cytokine IL-8 production in an enterotoxigenic B. fragilis (ETBF)-infected colonic epithelial cell (CEC) culture model. Compared to untreated infected cells, the addition of phage VA7 to ETBF-infected CECs led to significantly reduced bacterial counts and IL-8 levels. This in vitro study confirms the potential of phage VA7 as an antibacterial agent for use in prophylaxis or in the treatment of B. fragilis infections and associated colorectal carcinoma.


Assuntos
Bacteriófagos , Infecções por Bacteroides/terapia , Bacteroides fragilis/virologia , Terapia por Fagos , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Colo/patologia , Neoplasias Colorretais , Diarreia , Células Epiteliais , Humanos
6.
J Oral Pathol Med ; 49(7): 655-664, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32107794

RESUMO

BACKGROUND: Recent studies have demonstrated mesenchymal stem cell migration toward tumor locations. When applied locally, MSCs interact with the locally residing host cells. The mechanisms behind this are still unclear. We aimed to detect the possible action mechanisms of MSCs on the in vivo growth of primary human oral squamous cell carcinoma. METHODS: In mouse model of OSSC, chemotherapy with Cisplatin was done beginning from 9 day of tumor visualization. 3 weeks after tumor cell injection cultivated MSCs were administrated in tail vein or directly intra-tumorally. Animals underwent surveillance and afterward were sacrificed. Tumor growth was measured. MSCs biodistribution was assessed with bioluminescent analysis. Tumor tissues were tested morphologically and immunohistochemically for angiogenesis, hypoxia status, and cell apoptosis. RESULTS: In the group treated with Cisplatin in combination with mesenchymal stem cell injection, the average size of the tumor was 98.9 ± 7.65 mm3 . In the experimental group, tumor tissues were less outlined and the presence of necrotic areas and connective tissue basal layers was detected. Immunohistochemical surveys with CD31 and anti-carbonic anhydrase 9 demonstrated strongly developed micro-vessel structures and small isles of hypoxia in the tumor tissues. TUNEL assay revealed in the same group that tumor tissues were mostly comprised of apoptotic cells. Viable cell communities presented as small isles. CONCLUSION: The study demonstrates that intra-tumorally injected MSCs, combined with Cisplatin, leads to a minimal hypoxia status and increased apoptotic activity in tumor tissues, compared with the control group. This finding can be explained with better distribution of Cisplatin due to increased angiogenesis.


Assuntos
Carcinoma de Células Escamosas/terapia , Cisplatino/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Neoplasias Bucais/terapia , Animais , Apoptose , Movimento Celular , Humanos , Camundongos , Distribuição Tecidual
7.
Leukemia ; 34(1): 167-179, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31182781

RESUMO

Over the last few years, a detailed map of genetic and epigenetic lesions that underlie multiple myeloma (MM) has been created. Regulation of microRNA (miR)-dependent gene expression and mRNA splicing play significant roles in MM pathogenesis; however, to date an interplay between these processes is not yet delineated. Here we investigated miR-mediated regulation of splicing networks at the transcriptome level. Our studies show that a significant number (78%) of miRs which are either up- or down-regulated in patient CD138+ MM cells, but not in healthy donors (HD) CD138+ plasma cells (PC), target genes involved in early stages of pre-mRNA splicing. We also identified deregulated miRs that target core splicing factors (SF) and modifiers (SM, enhancers/silencers) which cause altered splicing in MM. Our studies suggest that Let-7f, in combination other miRs which are frequently and significantly deregulated in patients with overt MM, targets genes that regulate intron excision. Importantly, deregulated expression of certain miRs in MM promote increased intron retention, a novel characteristic of the MM genome, by inducing deregulated expression of the genes that regulate the splicing network. Our studies, therefore, provide the rationale for therapeutically targeting deregulated miRs to reverse aberrant splicing and improve patient outcome in MM.


Assuntos
MicroRNAs/genética , Mieloma Múltiplo/genética , Precursores de RNA/genética , Splicing de RNA/genética , Humanos
8.
Int J Surg Case Rep ; 53: 235-237, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30428439

RESUMO

INTRODUCTION: Gallbladder agenesis is a rare congenital abnormality with an incidence of 10-65 per 100,000. Approximately half of these patients are surgically operated on because of the symptoms similar to biliary colic, and correct diagnosis is established intra-operatively. We present a clinical case of gallbladder agenesis from our practice. PRESENTATION OF CASE: A 49 (forty-nine) - year- old women was admitted in the Emergency Department of our clinic. Symptoms were similar to the biliary colic. Ultrasonography showed hyperechogenic acoustic shadow on the projection of the gallbladder which was considered as constricted gallbladder and cholecystolithiasis was diagnosed. Laparoscopic cholecystectomy was considered. During laparoscopy gallbladder could not be found. Surgical operation was completed without conversion. Postoperative treatment included analgesics and antispasmodics. Pre-operative symptoms disappeared. One month later magnetic resonance cholangiopancreatography (MRCP) confirmed gallbladder agenesis diagnosis. Health condition of the patient is satisfactory, without any complications after a year of surgery. DISCUSSION: Gallbladder agenesis presented with symptoms similar to biliary colic can be diagnosed without surgical intervention. Conservative treatment consists of antispasmodic drugs. CONCLUSION: If the shrunken gallbladder is detected on the ultrasound, additional radiological examinations are required. MRCP is considered as a test of choice among the radiological investigations. If gallbladder agenesis is identified on laparoscopy, there is no need for further conversion. For postoperative follow up examination MRCP investigation is recommended.

9.
Oncol Lett ; 8(6): 2691-2694, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25364451

RESUMO

Benign osteoblastoma refers to a benign tumor of the bone. Osteoblastoma most commonly affects the vertebrae and long tubular bones, however, in rare cases is observed in the facial bones. The current study presents the case of a 12-year-old female patient with a tumor in the mandibular body. Radiological imaging revealed a lesion with regular contours. The lesion was radically resected and histological analysis of the specimen demonstrated features that are typical of a benign osteoblastoma. The consequential defects of the jaw were reconstructed using titanium implants and autologous bone transplantation. The patient remains disease free subsequent to a five-month follow-up period. The aim of the present report is to present a rare case of benign osteoblastoma of the mandible. This study demonstrated that correct diagnosis and complete surgical excision are important to reduce the risk of recurrence of a benign osteoblastoma.

10.
Pancreatology ; 12(4): 350-7, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22898637

RESUMO

BACKGROUND/OBJECTIVES: The small actin-binding protein destrin is one of the key regulators involved in remodeling of the actin cytoskeleton, a process crucial for cytokinesis, cell migration and polarized cell growth as well as for cancer cell migration and invasion. METHODS: A novel ex vivo nerve invasion model mirroring perineural cancer cell invasion as a key feature of pancreatic ductal adenocarcinoma has been previously established. Using this model, highly nerve-invasive clones of human pancreatic cancer cell lines have been obtained. Genome-wide transcriptional analyses of these cells revealed up-regulation of destrin in highly versus lowly nerve-invasive pancreatic cancer cells. RESULTS: Increased expression of destrin in these nerve-invasive cells was validated using quantitative RT-PCR and immunoblotting; concomitant changes in cell morphology were demonstrated using immunofluorescence analysis. Silencing of destrin by two specific siRNA oligonucleotides in Panc-1 pancreatic cancer cells decreased invasiveness and migration, and reduced proliferation of these cells. CONCLUSIONS: Destrin is upregulated in nerve-invasive pancreatic cancer cells and its expression might be related to perineural invasiveness.


Assuntos
Destrina/fisiologia , Invasividade Neoplásica/patologia , Sistema Nervoso/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Destrina/genética , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Regulação para Cima
11.
Mol Cancer ; 11: 19, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22471946

RESUMO

BACKGROUND: We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC) cells. METHODS: Syndecan-2 (SDC-2) expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling. RESULTS: SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides) did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and--further downstream--phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered. CONCLUSION: SDC-2 is a novel (perineural) invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.


Assuntos
Adenocarcinoma/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Pancreatite Crônica/metabolismo , Sindecana-2/metabolismo , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Pancreatite Crônica/genética , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Sindecana-2/genética
12.
Int J Cancer ; 129(10): 2349-59, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21207374

RESUMO

Glutamate has been implicated in tumorigenesis through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR). However, the function of a glutamate-to-AMPAR signal in pancreatic ductal adenocarcinoma (PDAC) has remained elusive. We now show that glutamate-mediated AMPA receptor activation increases invasion and migration of pancreatic cancer cells via activation of the classical MAPK pathway. Glutamate levels were increased in pancreatic cancer accompanied by downregulation of GluR subunits 1, 2, and 4. In pancreatic cancer precursor lesions, pancreatic intraepithelial neoplasia (PanIN), GluR1 subunit levels were strikingly and step-wise increased but its expression was rare in PDAC. Pharmacological inhibition or RNAi-mediated suppression of GluR1 or GluR2 did not affect cancer cell growth but significantly decreased invasion. In a K-ras wildtype cell line, AMPA receptor activation enhanced K-ras activity and--further downstream--phosphorylation of p38 and of p44/42. Preemptive blockade of AMPA receptors in a mouse model of pancreatic cancer inhibited tumor cell settling. AMPA receptor activation thus not only activates MAPK signalling but also directly increases activity of K-ras. Glutamate might serve as a molecular switch that decreases the threshold of K-ras-induced oncogenic signalling and increases the chance of malignant transformation of pancreatic cancer precursor lesions.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Ácido Glutâmico/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de AMPA/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Proto-Oncogênicas p21(ras)
13.
J Cell Mol Med ; 14(5): 1166-79, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19432821

RESUMO

Cell motility is controlled by the dynamic cytoskeleton and its related proteins, such as members of the ezrin/radixin/moesin (ERM) family, which act as signalling molecules inducing cytoskeleton remodelling. Although ERM proteins have been identified as important factors in various malignancies, functional redundancy between these proteins has hindered the dissection of their individual contribution. The aim of the present study was to analyse the functional role of moesin in pancreatic malignancies. Cancer cells of different malignant lesions of human and transgenic mice pancreata were evaluated by immunohistochemistry. For functional analysis, cell growth, adhesion and invasion assays were carried out after transient and stable knock-down of moesin expression in pancreatic cancer cells. In vivo tumourigenicity was determined using orthotopic and metastatic mouse tumour models. We now show that moesin knock-down increases migration, invasion and metastasis and influences extracellular matrix organization of pancreatic cancer. Moesin-regulated migratory activities of pancreatic cancer cells were in part promoted through cellular translocation of beta-catenin, and re-distribution and organization of the cytoskeleton. Analysis of human and different transgenic mouse pancreatic cancers demonstrated that moesin is a phenotypic marker for anaplastic carcinoma, suggesting that this ERM protein plays a specific role in pancreatic carcinogenesis.


Assuntos
Carcinoma/metabolismo , Carcinoma/patologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Proteínas dos Microfilamentos/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Animais , Caderinas/metabolismo , Carcinoma/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Fenótipo , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Adulto Jovem , beta Catenina/metabolismo
14.
Int J Oncol ; 35(5): 1111-6, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19787265

RESUMO

Perineural invasion of tumor cells is a characteristic feature of human pancreatic cancer. Unrevealing the molecular mechanisms that enable cancer cells to invade and grow along nerves is important for the development of novel therapeutic strategies in this disease. We have previously identified transcriptional changes in highly nerve invasive pancreatic cancer cells. Here we further analyzed one of the identified deregulated genes, MAPRE2, a microtubule-associated protein. MAPRE2 expression was significantly increased in high versus less nerve invasive pancreatic cancer cells, and changes of MAPRE2 expression resulted in altered actin distribution in these cells. MAPRE2 was predominately expressed in normal pancreatic acinar cells but absent in ductal cells. In pancreatic cancer, there was strong cytoplasmic and occasionally nuclear expression of MAPRE2 in the cancer cells themselves. Increased MAPRE2 mRNA levels in bulk pancreatic cancer tissues tended to be associated with reduced postoperative survival of pancreatic cancer patients. In conclusion, MAPRE2 is highly expressed in pancreatic cancer cells, and seems to be involved in perineural invasion. Therefore, targeting this microtubule-associated protein might be a promising approach in the therapy of pancreatic cancer.


Assuntos
Biomarcadores Tumorais/análise , Proteínas Associadas aos Microtúbulos/biossíntese , Invasividade Neoplásica/genética , Neoplasias Pancreáticas/patologia , Nervos Periféricos/patologia , Idoso , Western Blotting , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Proteínas Associadas aos Microtúbulos/genética , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Mol Cancer Ther ; 8(6): 1494-504, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19509238

RESUMO

Perineural invasion, the growth of tumor cells along nerves, is a key feature of pancreatic cancer. The cardinal symptom of pancreatic cancer, abdominal pain often radiating to the back, as well as the high frequency of local tumor recurrence following resection are both attributed to the unique ability of pancreatic tumor cells to invade the neuronal system. The molecular mechanisms underlying the neuroaffinity of pancreatic tumors are not completely understood. In this study, we developed a novel method to monitor ex vivo perineural invasion into surgically resected rat vagal nerves by different human pancreatic tumor cell lines. Genome-wide transcriptional analyses were employed to identify the consensus set of genes differentially regulated in all highly nerve-invasive (nerve invasion passage 3) versus less invasive (nerve invasion passage 0) pancreatic tumor cells. The critical involvement of kinesin family member 14 (KIF14) and Rho-GDP dissociation inhibitor beta (ARHGDIbeta) in perineural invasion was confirmed on RNA and protein levels in human pancreatic tumor specimens. We found significant up-regulation of KIF14 and ARHGDIbeta mRNA levels in patients with pancreatic cancer, and both proteins were differentially expressed in tumor cells invading the perineural niche of pancreatic cancer patients as detected by immunohistochemistry. Moreover, functional knockdown of KIF14 and ARHGDIbeta using small interfering RNA resulted in altered basal and/or perineural invasion of pancreatic tumor cells. Our work provides novel insights into the molecular determinants of perineural invasion in pancreatic cancer. The established nerve invasion model and the consensus signature of perineural invasion could be instrumental in the identification of novel therapeutic targets of pancreatic cancer as exemplified by KIF14 and ARHGDIbeta.


Assuntos
Movimento Celular/fisiologia , Perfilação da Expressão Gênica , Neoplasias Pancreáticas/genética , Nervo Vago/fisiologia , Animais , Anoikis , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Análise por Conglomerados , Regulação Neoplásica da Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Cinesinas/genética , Cinesinas/metabolismo , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
16.
Pancreatology ; 9(1-2): 165-72, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19077468

RESUMO

AIMS: Endocrine gland-derived vascular endothelial growth factor (EG-VEGF)/prokineticins have been identified as tissue-specific angiogenic factors. This study investigates the expression and localization of EG-VEGF and its receptors in pancreatic tissues and pancreatic stellate cells (PSCs). METHODS: mRNA levels of EG-VEGF/prokineticin 1 (PK1), prokineticin 2 (PK2) and their receptors 1 (PKR1) and 2 (PKR2) were measured in pancreatic tissues, pancreatic cancer cell lines and PSCs by quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR). Protein expression of PK1, PKR1 and PKR2 was assessed in pancreatic tissues by immunohistochemistry. Growth factor-induced secretion of EG-VEGF was measured by ELISA. RESULTS: QRT-PCR analysis in bulk tissues of normal pancreas, chronic pancreatitis and pancreatic ductal adenocarcinoma showed no significant difference of PK1 mRNA levels, whereas PK2 mRNA was barely detectable. High PK1 mRNA levels were observed only in cultured PSCs and microdissected islet cells, but not in cancer cells, and PK1 protein was localized mainly in islets and cancer-associated stromal cells. PKR1 and PKR2 proteins were present in endothelial cells of small blood vessels. TGF-beta(1) and PDGF-BB specifically stimulated PK1 secretion in PSCs. CONCLUSIONS: Islet and/or PSC-derived PK1 might act through its receptors on endothelial cells to increase angiogenesis in pancreatic diseases.


Assuntos
Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/biossíntese , Linhagem Celular Tumoral , Células Cultivadas , Hormônios Gastrointestinais/biossíntese , Humanos , Neuropeptídeos/biossíntese , Pâncreas/citologia , Pancreatite Crônica/metabolismo , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Receptores de Peptídeos/biossíntese , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo
17.
Clin Gastroenterol Hepatol ; 6(10): 1155-61, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18639493

RESUMO

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with an innate resistance to therapy. Pancreatic stellate cells (PSCs) produce this excessively desmoplastic microenvironment. The impact of PSC activity on PDAC behavior in vivo is analyzed. METHODS: 233 patients who underwent surgery for PDAC were evaluated by immunohistochemistry using antibodies against alpha-smooth muscle actin as a marker of PSC activity. Aniline was used to stain collagen deposition. The ratio of alpha-smooth muscle actin-stained area to collagen-stained area was defined as the activated stroma index (ASI). Survival analysis was performed using the Kaplan-Meier method. Prognostic factors were determined in a multivariable analysis using a Cox proportional hazards model. RESULTS: Four major patterns of collagen deposition were defined with regard to PSC activity. The combination of high stromal activity and low collagen deposition was associated with a worse prognosis, whereas the combination of high collagen deposition and low stromal activity indicated a better prognosis. Patients with the lowest ASI had the best median survival rate (25.7 mo). The highest ASI was found in patients with the worst median survival rate (16.1 mo; P = .007; lowest vs highest ASI: hazard ratio, 1.61; 95% confidence interval, 1.014-2.562). ASI was an independent prognostic marker in multivariable survival analysis comparable with the nodal status of cancer. CONCLUSIONS: The activated stroma index is a novel independent prognostic marker in PDAC in cases undergoing surgery. This finding highlights the impact of the microenvironment in cancer progression and on patient survival.


Assuntos
Actinas/análise , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patologia , Colágeno/análise , Idoso , Biomarcadores , Carcinoma Ductal Pancreático/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais
18.
Cancer Biol Ther ; 6(2): 218-27, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17224648

RESUMO

EMMPRIN (extracellular matrix metalloproteinase inducer, CD147) participates in the progression of various malignancies by stimulating the synthesis of specific matrix metalloproteinases (MMP) from peritumoral fibroblasts. In the present study, the expression and functional role of EMMPRIN was investigated in pancreatic neoplasm. QRT-PCR, immunohistochemistry, immunoblot, and ELISA analyses were used to analyze the expression, localization, and release of EMMRPIN. Silencing of EMMPRIN was performed using siRNA oligonucleotides, and functional consequences were assessed using growth assays, invasion assays, as well as MMP1/MMP2 and VEGF ELISA. EMMPRIN mRNA levels were 2.2-fold increased in pancreatic cancer (n = 52) and 2.0-3.5-fold increased in other pancreatic neoplasm (n = 105), but unchanged in chronic pancreatitis (n = 10) compared to normal pancreatic tissues (n = 9). Strong and predominantly membranous immunostaining was observed in the cancer cells and surrounding stromal cells. EMMPRIN serum levels were also significantly increased in pancreatic cancer patients (n = 44) (4.13 +/- 0.28 ng/ml) with an AUC of 0.97 compared to healthy volunteers (n = 29) (0.95 +/- 0.16 ng/ml; p < 0.0001) and with an AUC of 0.74 compared to chronic pancreatitis patients (n = 20) (2.98 +/- 0.5 ng/ml; p = 0.0021). EMMPRIN silencing did not significantly affect anchorage-dependent or -independent growth of pancreatic cancer cells. In contrast, EMMPRIN silencing in pancreatic stellate cells slightly repressed VEGF and MMP2 levels but strongly increased pro-MMP1 expression under coculture conditions. In conclusion, Increased EMMPRIN expression is present in different pancreatic neoplasm, likely representing a tumor-specific reaction with the potential to modulate the tumor microenvironment rather than a mere reflection of an activated stroma.


Assuntos
Basigina/biossíntese , Carcinoma Ductal Pancreático/metabolismo , Metaloproteases/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Western Blotting , Células/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Imuno-Histoquímica , Pâncreas/citologia , Pâncreas/metabolismo , Pancreatite Crônica/metabolismo
19.
J Clin Pathol ; 60(8): 885-95, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16775116

RESUMO

AIM: To determine the role of two antiapoptotic proteins of the inhibitor of apoptosis protein family, cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2), in human pancreatic carcinogenesis. METHODS: mRNA levels were measured in pancreatic tissues and pancreatic cancer cell lines by quantitative reverse transcriptase PCR. Protein expression was assessed in pancreatic cancer cell lines by immunoblotting and in pancreatic tissues by immunohistochemistry, and correlated with pathological and survival data. RESULTS: cIAP1 expression was constantly high in non-neoplastic pancreatic tissues, in pancreatic intraepithelial neoplasia (PanIN) lesions, as well as in a subset of primary and metastatic pancreatic ductal adenocarcinomas (PDAC), and a preferential cytoplasmatic localisation was observed in the tumour tissues. cIAP1 expression was rare in a cohort of cystic tumours. cIAP2 mRNA levels were significantly higher (2.4 fold) in PDAC than in normal tissues. cIAP2 protein was overexpressed in PDAC, and was detectable in low- and high-grade PanIN lesions. Moreover, cIAP2 was often expressed in pancreatic cystic tumours. cIAP1 and cIAP2 mRNA and protein were detected in all the examined cell lines. Survival analysis revealed a shorter survival in patients with cIAP1/cIAP2-positive tumours. CONCLUSIONS: cIAP1 might contribute to the regulation of the apoptotic process in the normal and in the neoplastic pancreas, depending on its subcellular localisation. Overexpression of cIAP2 is a common and early event in the progression of pancreatic cancer, and could therefore potentially influence the important pathophysiological aspects of PDAC, such as anoikis or chemoresistance.


Assuntos
Proteínas Inibidoras de Apoptose/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/química , Adenocarcinoma/patologia , Adulto , Idoso , Carcinoma in Situ/química , Carcinoma in Situ/patologia , Linhagem Celular Tumoral , Doença Crônica , Cistos/química , Citoplasma/química , Citoplasma/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica/métodos , Proteínas Inibidoras de Apoptose/análise , Masculino , Pessoa de Meia-Idade , Pâncreas/química , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/secundário , Pancreatite/genética , Pancreatite/patologia , RNA Mensageiro/análise , RNA Neoplásico/análise , Análise de Sobrevida
20.
Mol Cancer ; 4(1): 14, 2005 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-15817123

RESUMO

UNLABELLED: Hsulf-1 is a newly identified enzyme, which has the ability to decrease the growth of hepatocellular, ovarian, and head and neck squamous cell carcinoma cells by interfering with heparin-binding growth factor signaling. Since pancreatic cancers over-express a number of heparin-binding growth factors and their receptors, the expression and function of this enzyme in pancreatic cancer was analyzed. RESULTS: Pancreatic cancer samples expressed significantly (22.5-fold) increased Hsulf-1 mRNA levels compared to normal controls, and Hsulf-1 mRNA was localized in the cancer cells themselves as well as in peritumoral fibroblasts. 4 out of 8 examined pancreatic cancer cell lines expressed Hsulf-1, whereas its expression was below the level of detection in the other cell lines. Stable transfection of the Hsulf-1 negative Panc-1 pancreatic cancer cell line with a full length Hsulf-1 expression vector resulted in increased sulfatase activity and decreased cell-surface heparan-sulfate proteoglycan (HSPG) sulfation. Hsulf-1 expression reduced both anchorage-dependent and -independent cell growth and decreased FGF-2 mediated cell growth and invasion in this cell line. CONCLUSION: High expression of Hsulf-1 occurs in the stromal elements as well as in the tumor cells in pancreatic cancer and interferes with heparin-binding growth factor signaling.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Sulfotransferases/metabolismo , Adulto , Idoso , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Pâncreas/irrigação sanguínea , Pâncreas/citologia , Pâncreas/metabolismo , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Sulfatases/genética , Sulfotransferases/genética
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