cyclin-d1 and Pancreatic-Neoplasms

Intraductal papillary neoplasm of the bile duct (IPNB) is characterized by exophytic proliferation of neoplastic epithelial cells with fibrovascular stalks in bile duct lumen, mucin hypersecretion, and considerable dilatation or multilocular changes of the affected bile ducts. A mucin-producing bile duct tumor is an IPNB with excessive mucin production and clinical symptoms. Herein, the phenotypes as well as the tumorigenesis and progression of IPNB are reviewed with immunohistochemical assistance. The tumors are subdivided into three phenotypes: pancreatobiliary, intestinal, and gastric. About half of IPNB cases are of the pancreatobiliary type, and the remaining half are of the intestinal type. Aberrant expression of CDX2 with MUC2 and CK20 is related to the development of intestinal metaplasia. Inactivation of P16INK4a and nuclear expression of beta-catenin are related to the development of IPNB. Decreased expression of membranous beta-catenin and E-cadherin and aberrant expression of MMP-7 and -9 and of MUC1 are related to invasion of IPNB with tubular adenocarcinoma, whereas MUC2 is involved in the invasion of IPNB with mucinous carcinoma. IPNB can be regarded as a counterpart of intraductal papillary mucinous neoplasm (IPMN) of the pancreas, particularly the main duct type. More comparative studies between IPNB and pancreatic IPMN are recommended for further analysis of these papillary neoplasms.

Topics: Adenocarcinoma, Mucinous; beta Catenin; Bile Duct Neoplasms; Cadherins; Carcinoma, Ductal; Carcinoma, Papillary; CDX2 Transcription Factor; Cholangiocarcinoma; Cyclin D1; Disease Progression; Homeodomain Proteins; Humans; Immunohistochemistry; Keratin-20; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Mucin-1; Mucin-2; Mucins; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype

Topics: Animals; Apoptosis; Chelating Agents; Chromatography, High Pressure Liquid; Cyclin D1; Enzyme Inhibitors; Genes, erbB-2; Genes, p53; Genes, ras; Humans; Models, Chemical; Nucleic Acid Conformation; Pancreatic Neoplasms; Peptide Nucleic Acids; Peptides; Proto-Oncogene Proteins c-myc; Radioisotopes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Technetium; Tomography, Emission-Computed, Single-Photon

Many steps in the Wnt signalling pathway may be altered during the process of carcinogenesis. This Review focuses on the changes observed in gastrointestinal cancers. A literature search was undertaken and the currently available data summarised. Understanding the alterations to this signalling pathway may help to reveal future targets for therapeutic agents. In addition, since in some tumours, levels of components of the Wnt pathway have been found to correlate with clinical stage, their potential use as prognostic indicators is highlighted.

Topics: Axin Protein; beta Catenin; Carcinoma, Hepatocellular; Cyclin D1; Cytoskeletal Proteins; DNA-Binding Proteins; Frizzled Receptors; Gastrointestinal Neoplasms; Genes, APC; Genes, myc; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Matrix Metalloproteinase 7; Oligopeptides; Pancreatic Neoplasms; Prognosis; Proteins; Repressor Proteins; Signal Transduction; Trans-Activators; Wnt Proteins

Human neuroendocrine tumors exhibit unique biological properties, and defining the molecular genetic alterations that underlie these distinctive features remains an important challenge. In addition to the MEN1 tumor suppressor gene, the cyclin D1 oncogene has demonstrated a role in the pathogenesis of parathyroid and gastroenteropancreatic neuroendocrine tumors. Up-regulation of cyclin D1 is observed early in tumor formation, implying a possible role in tumor initiation. Overexpression of cyclin D1 in the parathyroid glands of mice resulted in the tandem regulation of cellular proliferation and hormonal secretion, a feature intrinsic to neuroendocrine tumors.

Topics: Cyclin D1; Gastrointestinal Neoplasms; Humans; Neuroendocrine Tumors; Pancreatic Neoplasms; Parathyroid Neoplasms

STMN2, as a key regulator in microtubule disassembly and dynamics, has recently been shown to participate in cancer development. However, the corresponding role in pancreatic ductal adenocarcinoma (PC), to our knowledge, has not been reported yet. In the current study, we systematically investigate the potential role of STMN2 in the progression of PC in vitro and vivo. Overexpression of STMN2 was prevalently observed in 81 human cases of PC tissues compared with that in the paired adjacent pancreas (54.3% vs 18.5%, P < 0.01), which was positively associated with multiple advanced clinical stages of PC patients (tumor size, T stage, lymph-node metastasis and the poor prognosis). Meanwhile, a close correlation between high STMN2 and cytoplasmic/nuclear β-catenin expression (P = 0.007) was observed in PC tissues and cell lines. STMN2 overexpression induced EMT and cell proliferation in vitro via stimulating EMT-like cellular morphology, cell motility and proliferation, and the change of EMT (Snail1, E-cadherin and Vimentin) and Cyclin D1 signaling. However, XAV939 inhibited STMN2 overexpression-enhanced EMT and proliferation. Conversely, KY19382 reversed STMN2 silencing- inhibited EMT and cell proliferation in vitro. Furthermore, activated STMN2 and β-catenin were co-localized in cytoplasm/nuclear in vitro. β-catenin/TCF-mediated the transcription of STMN2 by the potential binding sites (TTCAAAG). Finally, STMN2 promoted subcutaneous tumor growth following the activation of EMT and Cyclin D1 signaling. STMN2 overexpression promotes the aggressive clinical stage of PC patients and promotes EMT and cell proliferation in vitro and vivo. β-catenin/TCF-mediated the transcription of STMN2.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Stathmin; Wnt Signaling Pathway

Pancreatic cancer (PC) is an aggressive malignancy with poor prognosis. Accumulating studies have showed that long non-coding RNA (lncRNA) is a crucial regulator in various tumorigenesis and progression including PC. This research aims to explore the roles and molecular mechanism of lncRNA cancer susceptibility candidate 9 (CASC9) in PC.. The expression levels of lncRNA CASC9 and miR-497-5p were evaluated in PC tissues and paired adjacent healthy tissues by quantitative real-time PCR. PC cell lines were transfected with lentivirus targeting lncRNA CASC9, and cells proliferation, migration and invasion tests were conducted. Dual luciferase reporter assays were also carried out to explore the relationship between lncRNA CASC9, miR-497-5p and Cyclin D1 (CCND1).. LncRNA CASC9 was significantly up-regulated in PC tissues, while miR-497-5p expression was down-regulated. Down-regulation of lncRNA CASC9 in PC cells can significantly suppress the cell aggressiveness both in vitro and in vivo; moreover, knock-down of miR-497-5p could neutralize this impact. Additionally, the luciferase activity assay has assured that CCND1 was a downstream target of miR-497-5p.. LncRNA CASC9 can promote the PC progression by modulating miR-497-5p/CCND1 axis, which is potential target for PC treatment.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Pancreatic Neoplasms; RNA, Long Noncoding

Pancreatic cancer has the highest prevalence of cancer-associated cachexia among all cancers. ZIP4 promotes pancreatic cancer progression by regulating oncogenic miR-373, and perturbation of circular RNAs (circRNAs) is associated with cancer aggressiveness. This study aimed to identify circRNAs involved in ZIP4/miR-373-driven cancer growth and cachexia and decipher the underlying mechanism.. Differentially expressed circRNAs and potential targets of microRNA were identified through in silico analysis. The RNA interactions were determined by means of biotinylated microRNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. The function of circRNA in ZIP4-miR-373 signaling axis were examined in human pancreatic cancer cells, 3-dimensional spheroids and organoids, mouse models, and clinical specimens. Mouse skeletal muscles were analyzed by means of histology.. We identified circANAPC7 as a sponge for miR-373, which inhibited tumor growth and muscle wasting in vitro and in vivo. Mechanistic studies showed that PHLPP2 is a downstream target of ZIP4/miR-373. CircANAPC7 functions through PHLPP2-mediated dephosphorylation of AKT, thus suppressing cancer cell proliferation by down-regulating cyclin D1 and inhibiting muscle wasting via decreasing the secretion of transforming growth factor-β through STAT5. We further demonstrated that PHLPP2 induced dephosphorylation of CREB, a zinc-dependent transcription factor activated by ZIP4, thereby forming a CREB-miR-373-PHLPP2 feed-forward loop to regulate tumor progression and cancer cachexia.. This study identified circANAPC7 as a novel tumor suppressor, which functions through the CREB-miR-373-PHLPP2 axis, leading to AKT dephosphorylation, and cyclin D1 and transforming growth factor-β down-regulation to suppress tumor growth and muscle wasting in pancreatic cancer.

Topics: Animals; Cachexia; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Mice; MicroRNAs; Muscles; Pancreatic Neoplasms; Phosphoprotein Phosphatases; Proto-Oncogene Proteins c-akt; RNA, Circular; Transforming Growth Factor beta

Semaphorins (SEMAs) are axon guidance factors that participate in axonal connections and nerve system development. However, the functional roles of SEMAs in tumorigenesis are still largely uncovered. By using in silico data analysis, we found that SEMA6C was downregulated in pancreatic cancer, and its reduction was correlated with worse survival rates. RNA sequencing revealed that cell cycle-related genes, especially cyclin D1, were significantly altered after blockage of SEMA6C by neutralizing antibodies or ectopic expressions of SEMA6C. Mechanistic investigation demonstrated that SEMA6C acts as a tumor suppressor in pancreatic cancer by inhibiting the AKT/GSK3 signaling axis, resulting in a decrease in cyclin D1 expression and cellular proliferation. The enhancement of cyclin D1 expression and cyclin-dependent kinase activation in SEMA6C-low cancer created a druggable target of CDK4/6 inhibitors. We also elucidated the mechanism underlying SEMA6C downregulation in pancreatic cancer and demonstrated a novel regulatory role of miR-124-3p in suppressing SEMA6C. This study provides new insights of SEMA6C-mediated anti-cancer action and suggests the treatment of SEMA6C-downregulated cancer by CDK4/6 inhibitors.

Topics: beta Catenin; Catenins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Pancreatic Neoplasms; Proto-Oncogene Proteins c-akt; Semaphorins

Anti-silencing function 1B (ASF1B) has been reported to be associated with the occurrence of many kinds of tumors. However, the biological effect and action mechanism of ASF1B in pancreatic cancer (PC) tumorigenesis remain unclear. The expression and prognosis value of ASF1B in PC were analyzed using GEPIA, GEO, and Kaplan-Meier plotter databases. The diagnostic value of ASF1B in PC was determined by receiver operating characteristic curve. The relationship between ASF1B expression and the clinical feathers in PC was investigated based on TCGA. qRT-PCR and western blot analyses were used to measure ASF1B expression in PC cells. Cell proliferation was evaluated by MTT and EdU assays, and apoptosis was examined by TUNEL and caspase-3 activity assays. Western blot analysis was utilized to detect the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, Bax, Bcl-2, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling proteins. ASF1B was overexpressed in several digestive cancers, including PC. Upregulated ASF1B was correlated with the poor prognosis and clinical features in PC patients. The area under the curve (AUC) value of ASF1B was 0.990. ASF1B was also overexpressed in PC cells. ASF1B silencing inhibited PC cell proliferation, promoted apoptosis, and increased caspase-3 activity, which were accompanied by the reduction of PCNA and cyclin D1 expression and increase of the ratio of Bax/Bcl-2 expression. Additionally, ASF1B silencing suppressed the PI3K/Akt pathway and 740Y-P treatment partially abolished the effects of ASF1B knockdown on PC cells. In conclusion, ASF1B silencing retarded proliferation and promoted apoptosis in PC cells by inactivation of the PI3K/Akt pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinogenesis; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Signal Transduction

Pancreatic cancer will be positioned by the year 2030 as the second cause of oncological death after lung cancer. The pathophysiology of the most common variety, which involves the adenocarcinoma of the pancreas, represents one of the main challenges for current oncology to explain its tumorigenesis and create a targeted treatment. The tumor microenvironment, metastatic capacity, and lack of early diagnosis lead patients to present advanced stages at the time of diagnosis. Despite numerous efforts, little progress has been made in clinical outcomes and with respect to the improved survival of these patients. For this reason, in recent years, numerous diagnostic tests, treatments, and possible approaches in the fields of radiotherapy, chemotherapy, immunotherapy, and surgery have been developed to find a combination of methods that improves life expectancy in patients diagnosed with this disease. On the other hand, the scientific community has made numerous advances in the molecular bases of pancreatic cancer since several oncogenetic pathways have been described and the markers expressed by the tumor have proven to be useful in the prognosis of pancreatic adenocarcinoma. These molecular alterations allow the study of possible therapeutic targets that improve the prognosis of these patients, but even numerous tumor cell-individual interactions must be explained to understand the underlying pathophysiology causing the high mortality. Therefore, the purpose of our study is to examine the expression of markers such as EGFR, Cyclin D1, andCDK4 in order to find a relationship with the possible long-term prognostic factors of patients affected by pancreatic ductal adenocarcinoma. Our results show that there is a prognostic role for ErbB2, EGFR, beta catenin, cyclin D1, and CDK4. Of these, we highlight the clinical importance of ErbB2 in the survival rates of patients who overexpress this component.

Topics: Adenocarcinoma; Carcinoma, Pancreatic Ductal; Cyclin D1; Humans; Pancreatic Neoplasms; Receptor, ErbB-2; Tumor Microenvironment

Silencing of the Apoptosis associated Tyrosine Kinase gene (AATK) has been described in cancer. In our study, we specifically investigated the epigenetic inactivation of AATK in pancreatic adenocarcinoma, lower grade glioma, lung, breast, head, and neck cancer. The resulting loss of AATK correlates with impaired patient survival. Inhibition of DNA methyltransferases (DNMTs) reactivated AATK in glioblastoma and pancreatic cancer. In contrast, epigenetic targeting via the CRISPR/dCas9 system with either EZH2 or DNMT3A inhibited the expression of AATK. Via large-scale kinomic profiling and kinase assays, we demonstrate that AATK acts a Ser/Thr kinase that phosphorylates TP53 at Ser366. Furthermore, whole transcriptome analyses and mass spectrometry associate AATK expression with the GO term 'regulation of cell proliferation'. The kinase activity of AATK in comparison to the kinase-dead mutant mediates a decreased expression of the key cell cycle regulators Cyclin D1 and WEE1. Moreover, growth suppression through AATK relies on its kinase activity. In conclusion, the Ser/Thr kinase AATK represses growth and phosphorylates TP53. Furthermore, expression of AATK was correlated with a better patient survival for different cancer entities. This data suggests that AATK acts as an epigenetically inactivated tumor suppressor gene.

Topics: Adenocarcinoma; Apoptosis Regulatory Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Tumor Suppressor Protein p53

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated β-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

Topics: Adenocarcinoma; Animals; Apelin; Apelin Receptors; beta Catenin; Carcinoma, Pancreatic Ductal; Cyclin D1; Glucose; Humans; Mice; Oncogenes; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, G-Protein-Coupled; Signal Transduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors has one of the worst prognoses, and the role of long noncoding RNAs (lncRNAs) in the biological and pathological processes of pancreatic cancer, including tumor cell proliferation, is a popular topic in tumor research. Our previous study revealed the correlation between high levels of the lncRNA-SOX2OT (SOX2OT) with poor survival outcomes. Cell Counting Kit-8, EdU, Flow cytometry and Colony formation assays as well as Xenograft growth of PDAC cells in mice were used for the detection of PDAC cells proliferation progression. Fluorescence in situ hybridization, RNA-binding protein pulldown and RNA immunoprecipitation assays were also used to identify the putative mechanisms of SOX2OT participating in the tumor progression. SOX2OT and its potential downstream targets were verified by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). SOX2OT was confirmed to promote the proliferation of PDAC cells. It was found to directly physically bind to FUS and we also demonstrated that FUS protein stability was affected by binding with SOX2OT and FUS could suppressed PDAC tumor by regulating cell cycle-associated factors CCND1 and p27. Our findings suggest that SOX2OT may act as a tumor promoter in PDAC through physically binding FUS and regulating its downstream cell cycle-associated factors CCND1 and p27. It may serve as an effective target for antitumor treatment for pancreatic cancer.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Heterografts; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Pancreatic Neoplasms; RNA-Binding Protein FUS; RNA, Long Noncoding; RNA, Messenger

Gemcitabine is clinically used to treat certain types of cancers, including pancreatic and biliary cancer. We investigated the signal transduction pathways underlying the local antitumor effects of gemcitabine-eluting membranes (GEMs) implanted in pancreatic/biliary tumor-bearing nude mice. Here, we report that GEMs increased the E3 ubiquitin ligase c-CBL protein level, leading to degradation of epidermal growth factor receptor (EGFR) in SCK and PANC-1 cells. GEMs decreased the RAS and PI3K protein levels, leading to a reduction in the protein levels of active forms of downstream signaling molecules, including PDK, AKT, and GSK3β. GEM reduced proliferation of cancer cells by upregulating cell cycle arrest proteins, particularly p53 and p21, and downregulating cyclin D1 and cyclin B. Moreover, GEMs reduced the levels of proangiogenic factors, including VEGF, VEGFR2, CD31, and HIF-1α, and inhibited tumor cell migration and invasion by inducing the expression of E-cadherin and reducing that of N-cadherin, snail, and vimentin. We demonstrated that local delivery of gemcitabine using GEM implants inhibited tumor cell growth by promoting c-CBL-mediated degradation of EGFR and inhibiting the proliferation, angiogenesis, and epithelial-mesenchymal transition of pancreatic/biliary tumors. Use of gemcitabine-eluting stents can improve stent patency by inhibiting the ingrowth of malignant biliary obstructions.

Topics: Animals; Antigens, CD; Cadherins; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Cyclin B; Cyclin D1; Deoxycytidine; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gemcitabine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Pancreas; Pancreatic Neoplasms; Platelet Endothelial Cell Adhesion Molecule-1; Proto-Oncogene Proteins c-cbl; Signal Transduction; Ubiquitin-Protein Ligases; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Quercetin, an abundant flavonoid found in various fruits and vegetables, displays multiple biological activities, including anticancer effects. Therefore, quercetin is receiving increasing attention as a potential adjuvant anticancer treatment. Gemcitabine (GEM) resistance is a major issue for clinicians and patients with advanced cancers, making it crucial to determine ways to bolster its effects. In this study, we explored the anticancer effects and mechanistic actions of quercetin in GEM-resistant cancer cells. Pancreatic cancer (BxPC-3, PANC-1) and hepatocellular carcinoma (HepG2, Huh-7) cell lines were studied. Proliferation assays showed that quercetin had cytotoxic effects on GEM-resistant cell lines (HepG2 and PANC-1), and flow cytometric analysis indicated a significant pro-apoptotic effect on these cell lines. GEM treatment, in combination with quercetin, resulted in increased anticancer effects compared with GEM alone. Quercetin led to S phase arrest in GEM-resistant cell lines, and western blot analysis revealed tumour protein p53 upregulation and cyclin D1 downregulation. This study provides mechanistic insight into the anticancer effects of quercetin and suggests that quercetin adjuvant treatment may benefit patients who are resistant to GEM therapy.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Deoxycytidine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Gemcitabine; Hep G2 Cells; Humans; Liver Neoplasms; Pancreatic Neoplasms; Quercetin; Tumor Suppressor Protein p53

Topics: Angelica; Antineoplastic Agents; Apoptosis; Benzopyrans; Butyrates; Caspase 3; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Cyclin D1; Cyclin-Dependent Kinase 4; G1 Phase Cell Cycle Checkpoints; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Phosphorylation; Plant Extracts; Plant Roots

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)-3β, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine-sensitive BxPC-3 cell-derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3β-specific inhibitor alone attenuated the viability and proliferation of the gemcitabine-resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine-resensitizing effect of GSK3β inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro-oncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Deoxycytidine; Drug Resistance, Neoplasm; E2F1 Transcription Factor; Gemcitabine; Glycogen Synthase Kinase 3 beta; Heterografts; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Phosphorylation; Retinoblastoma Protein; Ribonucleoside Diphosphate Reductase; Thymidine Kinase; Thymidylate Synthase; Transcription, Genetic

C6orf106 was highly expressed in lung and breast cancer, and proposed as clinicopathologic factor for the development of those types of cancer. However, its expression in pancreatic cancer and the mechanism that C6orf106 functions as an oncogene has not been confirmed. In the present study, we found that C6orf106 was also up-regulated in pancreatic cancer tissues and cell lines. Furthermore, C6orf106 expression was associated with advanced T stage (P = 0.010), positive regional lymph node metastasis (P = 0.012), and advanced TNM stage (P = 0.006). In vitro experiments also showed that C6orf106 served a tumor enhancer in pancreatic cancer, through increasing the expression of Snail, Cyclin D1 and Cyclin E1, and reducing the expression of E-cadherin via activating extracellular-signal-regulated kinase (ERK)- p90-kDa ribosomal S6 kinases (P90RSK) signaling pathway. The addition of ERK inhibitor PD98059 counteracted the upregulation of Snail, Cyclin D1 and Cyclin E1, and restored the expression of E-cadherin, which indicated that C6orf106 was an upstream factor of ERK signaling pathway. Taken together, the present study indicates that C6orf106 facilitates invasion and proliferation of pancreatic cancer cells, likely via activating ERK-P90RSK signaling pathway.

Topics: Adult; Aged; Cadherins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Neoplasm Proteins; Oncogene Proteins; Pancreatic Neoplasms; Signal Transduction; Snail Family Transcription Factors

Multiple previous studies have demonstrated that the dysregulation of microRNAs (miRNAs) is implicated in the occurrence and development of pancreatic cancer. Therefore, a further characterisation of deregulated miRNAs in pancreatic cancer may provide novel insight into the oncogenesis and progression of pancreatic cancer, which may facilitate the identification of effective therapeutic targets for treating patients with this disease. In the present study, reverse transcription‑quantitative polymerase chain reaction analysis demonstrated that the expression level of miRNA‑584‑5p (miR‑584) was significantly decreased in pancreatic cancer tissues and cell lines. It was demonstrated that restoration of miR‑584 expression significantly suppressed the proliferative and invasive ability of pancreatic cancer cells. Bioinformatics analysis predicted that cyclin D1 (CCND1) was a putative target of miR‑584. Subsequent experiments demonstrated that CCND1 was a direct target gene of miR‑584 in pancreatic cancer cells. Furthermore, the inhibition of CCND1 mimicked the suppressive effect of miR‑584 overexpression in pancreatic cancer cells. The restoration of CCND1 expression significantly abolished the inhibitory effects of miR‑584 overexpression on pancreatic cancer cells. Collectively, the present results demonstrated that miR‑584 inhibited the development of pancreatic cancer by directly targeting CCND1, suggesting that this miRNA may represent a potential therapeutic target for this fatal disease.

Topics: Adult; Aged; Apoptosis; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Pancreatic Neoplasms; Prognosis; Tumor Cells, Cultured

It is well-known that the activation status of the P53, signal transducer and activator of transcription (Stat)3 and nuclear factor (NF)‑κB signaling pathways determines the radiosensitivity of cancer cells. However, the function of these pathways in radiosensitive vs radioresistant cancer cells remains elusive. The present study demonstrated that adaptive expression of epidermal growth factor (EGF) following exposure to ionizing radiation (IR) may induce radiosensitization of pancreatic cancer (PC) cells through induction of the cyclin D1/P53/poly(ADP‑ribose) polymerase pathway. By contrast, adaptively expressed interleukin (IL)‑6 and insulin‑like growth factor (IGF)‑1 may promote radioresistance of PC cells, likely through activation of the Stat3 and NF‑κB pathways. In addition, cyclin D1 and survivin, which are specifically expressed in the G1/S and G2/M phase of the cell cycle, respectively, are mutually exclusive in radiosensitive and radioresistant PC cells, while Bcl‑2 and Bcl‑xL expression does not differ between radiosensitive and radioresistant PC cells. Therefore, adaptively expressed EGF and IL‑6/IGF‑1 may alter these pathways to promote the radiosensitivity of PC cancers. The findings of the present study highlight potential makers for the evaluation of radiosensitivity and enable the development of effective regimens for cancer radiotherapy.

Topics: Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cyclin D1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Poly (ADP-Ribose) Polymerase-1; Radiation Tolerance; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation

Pancreatic cancer has a 5-year survival rate below 10% and the treatment options are limited. Signal transducer and activator of transcription (STAT3) is a constitutively expressed protein in human pancreatic cancers and is associated with their poor prognosis. Targeting of STAT3 signaling using novel therapeutic agents is a potential strategy for pancreatic cancer treatment. Diarylidenylpiperidone (DAP) compounds, such as H-4073 and HO-3867, have been shown to be STAT3 inhibitors in several human ovarian cancers. Particularly, HO-3867 is an N-hydroxypyrroline derivative of DAP that has targeted cytotoxicity toward cancer cells without affecting healthy cells. In the present study, we evaluated the anticancer efficacy of H-4073 and HO-3867 in a human pancreatic cell line (AsPC-1). We found that both the compounds exhibited potential cytotoxicity to AsPC-1 cells by inducing G2/M cell-cycle arrest, apoptosis, and cell death, by mitochondrial damage and inhibition of STAT3 phosphorylation. In summary, H-4073 and HO-3867 are cytotoxic to AsPC-1 cells and seem to act through similar mechanisms, including STAT3 inhibition, cell-cycle arrest, and apoptosis.

Topics: Apoptosis; Biomarkers, Tumor; Cell Line, Tumor; Cyclin D1; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; Mitochondria; Pancreatic Neoplasms; Phosphorylation; Piperidones; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; STAT3 Transcription Factor

Despite improvements in surgical procedures and comprehensive therapies, pancreatic cancer remains one of the most aggressive and deadly human malignancies. It is therefore necessary to determine which cellular mediators associate with prognosis in pancreatic cancer so as to improve the treatment of this disease. In the present study, mRNA array and immunohistochemical analyses showed that KLF5 is highly expressed in tissue samples from three short-surviving patients with pancreatic cancer. Survival analysis using data from The Cancer Genome Atlas showed that patients highly expressing KLF5 exhibited shorter overall and tumor-free survival times. Mechanistically, KLF5 promoted expression of E2F1, cyclin D1 and Rad51, while inhibiting expression of p16 in pancreatic cancer cells. Finally, flow cytometric analyses verified that KLF5 promotes G1/S progression of the cell cycle in pancreatic cancer cells. Collectively, these findings demonstrate that KLF5 is an important prognostic biomarker in pancreatic cancer patients, and they shed light on the molecular mechanism by which KLF5 stimulates cell cycle progression in pancreatic cancer.

Topics: Aged; Aged, 80 and over; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; E2F1 Transcription Factor; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Male; MicroRNAs; Middle Aged; Pancreatic Neoplasms; Prognosis; Rad51 Recombinase; Survival Analysis; Transcriptome

About 8384 cases of solid pseudopapillary neoplasms (SPN) of pancreas have been published in English literature, from 1933 to 2018. This is a low-grade tumor that usually occurs in children but is rare in adults and, in exceptional cases, can show extrapancreatic localization. In this paper we present 2 unusual cases of SPNs, 1 with retroperitoneal location (case 1) and 1 that was firstly diagnosed as a G1 neuroendocrine tumor (NET) and showed hepatic metastases after 13 years (case 2).. No symptoms in first case. The tumor was incidentally diagnosed, during ultrasound examination. In the second case, the metastasis was observed during regular follow-up.. The diagnosis was established based on the histological features and immunohistochemical profile that showed positivity for vimentin, nuclear β-catenin, cyclin D1, CD10, and SRY-related high-mobility group box 11 and negativity for maspin.. Surgical excision, in both cases.. No recurrences in first case, at 5 months after diagnosis. Hepatic metastases in the second case, at 13 years after diagnosis, with portal invasion after another 15 months.. Without a complex immunoprofile, SPN can be misdiagnosed as NET. SPN can be a low-grade tumor but long-time follow-up is mandatory to detect delayed metastases. A correct diagnosis is necessary for a proper therapeutic management.

Topics: Adenocarcinoma, Papillary; Adult; beta Catenin; Biomarkers, Tumor; Cyclin D1; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasms, Cystic, Mucinous, and Serous; Neprilysin; Neuroendocrine Tumors; Pancreas; Pancreatectomy; Pancreatic Neoplasms; Prognosis; Treatment Outcome; Vimentin

The ETS family transcription factor ETV4 is aberrantly expressed in a variety of human tumors and plays an important role in carcinogenesis through upregulation of relevant target gene expression. Here, it is demonstrated that ETV4 is overexpressed in pancreatic cancer tissues as compared with the normal pancreas, and is associated with enhanced growth and rapid cell-cycle progression of pancreatic cancer cells. ETV4 expression was silenced through stable expression of a specific short hairpin RNA (shRNA) in two pancreatic cancer cell lines (ASPC1 and Colo357), while it was ectopically expressed in BXPC3 cells. Silencing of ETV4 in ASPC1 and Colo357 cells reduced the growth by 55.3% and 38.9%, respectively, while forced expression of ETV4 in BXPC3 cells increased the growth by 46.8% in comparison with respective control cells. Furthermore, ETV4-induced cell growth was facilitated by rapid transition of cells from G

Topics: Adenovirus E1A Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Pancreatic Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Transcription, Genetic; Up-Regulation

The pathogenesis and etiology of pancreatic cancer remain to be fully elucidated; therefore, associated investigations are required to improve the outcome and prognosis of patients. In the present study, the effects of the overexpression of p16ink4a on the Wnt/β‑catenin signaling pathway were investigated in pancreatic cancer cell lines. Two pancreatic cancer cell lines, Bxpc‑3 and Miapaca‑2, characterized by low expression of p16ink4a, were transfected with the pc‑DNA3.0‑p16ink4a plasmid. After 24 h, Reverse transcription‑polymerase chain reaction and western blot analyses were performed to evaluate the expression of p16ink4a, β‑catenin, which is a key molecule in the Wnt/β‑catenin signaling pathway, c‑myc and cyclin D1, which are molecules downstream of β‑catenin. The expression of p16ink4a was significantly upregulated in the transfected cells. Consequently, the expression of β‑catenin was inhibited, whereas the expression levels of c‑myc and cyclin D1 were not altered significantly. The increased expression of p16ink4a may affect the activity of Wnt/β‑catenin signaling through modulation of the expression of β‑catenin. The results of the present study provide information for the future development of targeted treatments for pancreatic cancer.

Topics: beta Catenin; Biomarkers; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression; Genes, myc; Humans; Pancreatic Neoplasms; RNA, Messenger; Wnt Signaling Pathway

A specific expression of miRNA in pancreatic cancer renders it the novel diagnostic marker of pancreatic cancer. Therefore, we investigated how the anticancer effect of miRNA‑27a suppressed cell growth and induced apoptosis of human pancreatic cancer cells. We upregulated miRNA‑27a expression in PANC-1 cells using miRNA‑27a mimic, which demonstrated that induction of cell growth and suppression of apoptosis of human pancreatic cancer cells were observed. However, anti‑miRNA‑27a inhibited cell growth and apoptosis in pancreatic cancer cells. The downregulation of miRNA‑27a suppressed Wnt/β-catenin pathway. The inhibition of Wnt/β-catenin pathway increased the anticancer effects of anti‑miRNA‑27a on human pancreatic cancer cells. Taken together, miRNA‑27a promotes the proliferation and inhibits apoptosis of human pancreatic cancer cells via Wnt/β-catenin pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Pancreatic Neoplasms; Up-Regulation; Wnt Signaling Pathway

The overall 5-year survival rate of patients with human pancreatic cancer remains less than 8% because of its aggressive growth, early metastasis and resistance to conventional chemoradiotherapy. It is essential to develop innovative and effective therapeutic agents to improve its prognosis. Demethylzeylasteral (ZST93) is a novel triterpenoid monomer extracted from the xylem of Tripterygium roots. Our study aimed to assess the effects of ZST93 on cell proliferation and its role in the chemosensitivity to gemcitabine in human pancreatic cancer cells. The effects of ZST93 on cancer cell proliferation, cell cycle distribution, apoptosis and autophagy were evaluated in various human pancreatic cancer cell lines, and the antitumor effects of ZST93 alone and in combination with gemcitabine were identified in a xenograft mouse model. The results showed that ZST93 could inhibit the proliferation of pancreatic cancer cells and arrest cell cycle at G0/G1 phase by regulating the expression of Cyclin D1 and Cyclin A2. Moreover, ZST93 killed pancreatic cancer cells through two different mechanisms: inducing autophagic cell death at low concentrations and apoptotic cell death at high concentrations. Furthermore, ZST93 could enhance the chemosensitivity of pancreatic cancer cells to gemcitabine both in vitro and in vivo through modulation of the cross talk between autophagy and apoptosis. ZST93 is a potential therapeutic agent for developing novel therapeutic strategies in human pancreatic cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Cell Growth Processes; Cell Line, Tumor; Cyclin A2; Cyclin D1; Deoxycytidine; Drug Synergism; G1 Phase Cell Cycle Checkpoints; Gemcitabine; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Random Allocation; RNA, Messenger; Triterpenes; Xenograft Model Antitumor Assays

Low 5-year survival rates, increasing incidence, as well as the specific challenges of targeting pancreatic cancer, clearly support an urgent need for new multifunctional drugs for the prevention and treatment of this fatal disease. Natural products, such as abietane-type diterpenoids, are widely studied as promiscuous anticancer agents. In this study, dehydroabietic oximes were identified as potential compounds to target pancreatic cancer and cancer-related inflammation. The compounds inhibited the growth of human pancreatic cancer Aspc-1 cells with IC

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Nitric Oxide; Oximes; Pancreatic Neoplasms; Ribosomal Protein S6 Kinases, 90-kDa

The ω3-polyunsaturated fatty acid docosahexenoic acid (DHA) is known to induce apoptosis of cancer cells. In this study, DHA was shown to reduce viability of pancreatic cancer cells (PANC-1) by inducing DNA fragmentation, activating caspase-3, and increasing the ratio of Bax/Bcl-2. To determine the DHA mechanism of action, the impact of DHA on the activation of the key signaling proteins epidermal growth factor receptor (EGFR), signal transducer and activator of transcription factor 3 (STAT3), nuclear transcription factor-κB (NF-κB), and IκBα in PANC-1 cells was probed. The observed DHA suppression of NF-κB DNA-binding activity was found to result from reduced IκBα phosphorylation. The observed DHA-induced suppression of STAT3 activation was found to be the result of suppressed EGFR activation, which derives from the inhibitory effect of DHA on the integrity of localization of EGFR to cell membrane lipid rafts. Since the activation of STAT3 and NF-κB mediates the expression of survival genes cyclin D1 and survivin, DHA induced apoptosis by suppressing the STAT3/NF-κB-cyclin D1/survivin axis. These results support the proposal that DHA-induced apoptosis of pancreatic cells occurs via disruption of key pro-cell survival signaling pathways. We suggest that the consumption of DHA-enriched foods could decrease the incidence of pancreatic cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Cyclin D1; DNA Fragmentation; Docosahexaenoic Acids; Dose-Response Relationship, Drug; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Membrane Microdomains; NF-kappa B; Pancreatic Neoplasms; RNA, Messenger; STAT3 Transcription Factor; Survivin

Pancreatic cancer is highly malignant and characterised by rapid and uncontrolled growth. While some of the important regulatory networks involved in pancreatic cancer have been determined, the cancer relevant genes have not been fully identified.. We screened genes that may control proliferation in pancreatic cancer in seven pairs of matched pancreatic cancer and normal pancreatic tissue samples. We examined KIF15 expression in pancreatic cancer tissues and the effect of KIF15 on cell proliferation in vitro and in vivo. The mechanisms underlying KIF15 promotion of cell proliferation were investigated.. mRNA microarray and functional analysis identified 22 genes that potentially play an important role in the proliferation of pancreatic cancer. High-content siRNA screening evaluated whether silencing these 22 genes affected proliferation of pancreatic cancer. Notably, silencing KIF15 exhibited the most potent inhibition of proliferation compared with the rest of the 22 genes. KIF15 was upregulated in human pancreatic cancer tissues, and higher KIF15 expression levels correlated with shorter patient survival times. Upregulation KIF15 promoted pancreatic cancer growth. KIF15 upregulated cyclin D1, CDK2, and phospho-RB and also promoted G1/S transition in pancreatic cancer cells. KIF15 upregulation activated MEK-ERK signalling by increasing p-MEK and p-ERK levels. MEK-ERK inhibitors successfully inhibited cell cycle progression, and PD98059 blocked KIF15-mediated pancreatic cancer proliferation in vivo and in vitro.. This study identified KIF15 as a critical regulator that promotes pancreatic cancer proliferation, broadening our understanding of KIF15 function in tumorigenesis.

Topics: Aged; Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Kinesins; Male; MAP Kinase Signaling System; Mice; Middle Aged; Pancreatic Neoplasms; Protein Kinase Inhibitors; Xenograft Model Antitumor Assays

Pancreatic cancer is the fourth leading cause of cancer‑associated deaths in Western countries, and ranks sixth among cancer‑associated diseases, with the highest mortality rate in China. Deregulation of micro (miR) RNA may contribute to the occurrence and progression of numerous cancers, including pancreatic cancer. In particular, deregulation of microRNA‑720 (miR‑720) has been reported in various types of human cancer. However, the expression and biological role of miR‑720 in pancreatic cancer remains to be elucidated. The present study aimed to investigate the expression and functional role of miR‑720 in pancreatic cancer and determine the underlying regulatory mechanism. The results demonstrated that miR‑720 was expressed at low levels in pancreatic cancer tissue samples and cell lines. Upregulating miR‑720 suppressed pancreatic cancer cell proliferation and invasion in vitro. Additionally, cyclin D1 (CCND1) was identified as the direct target gene of miR‑720 in pancreatic cancer. Furthermore, CCND1 was significantly upregulated in pancreatic cancer tissues and inversely correlated with miR‑720 expression. Furthermore, CCND1 re‑expression partially abrogated the inhibitory effects of miR‑720 on pancreatic cancer cells. Overall, miR‑720 may act as a tumor suppressor by directly targeting CCND1 in pancreatic cancer.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Pancreatic Neoplasms; RNA Interference

Recent studies suggest that progesterone may possess anti-tumorigenic properties. However, a growth-modulatory role of progestins in human cancer cells remains obscure. With the discovery of a new class of membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor gene family, it becomes important to study the effect of this hormone on proliferation of tumor cells that do not express classical nuclear progesterone receptors (nPRs). To identify a cell line expressing high levels of mPRs and lacking nPRs, we examined mRNA levels of nPRs and three forms of mPRs in sixteen human tumor cell lines of different origin. High expression of mPR mRNA has been found in pancreatic adenocarcinoma BxPC3 cells, while nPR mRNA has not been detected in these cells. Western blot analysis confirmed these findings at the protein level. We revealed specific binding of labeled progesterone in these cells with affinity constant similar to that of human mPR expressed in yeast cells. Progesterone at high concentration of 20 μM significantly reduced the mRNA levels of proliferation markers Ki67 and PCNA, as well as of cyclin D1, and increased the mRNA levels of cyclin dependent kinase inhibitors p21 and p27. Progesterone (1 μM and 20 μM) significantly inhibited proliferative activity of BxPC3 cells. These results point to anti-proliferative effects of the progesterone high concentrations on BxPC3 cells and suggest that activation of mPRs may mediate this action. Our data are a starting point for further investigations regarding the application of progesterone in pancreatic cancer.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Jurkat Cells; Ki-67 Antigen; MCF-7 Cells; Pancreatic Neoplasms; Progesterone; Proliferating Cell Nuclear Antigen; Receptors, Progesterone

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies. The Yes-associated protein-1 (YAP) plays a critical role in cell proliferation, apoptosis and angiogenesis. Verteporfin is a photosensitizer used in photodynamic therapy and also a small molecular inhibitor of the Hippo-YAP pathway. However, little is known about whether verteporfin could inhibit YAP activity in PDAC cells. Our present results showed that verteporfin suppressed the proliferation of human PDAC PANC-1 and SW1990 cells by arresting cells at the G1 phase, and inducing apoptosis in dose- and time-dependent manners. Verteporfin also inhibited the tumor growth on the PDAC xenograft model. Treatment with verteporfin led to downregulation of cyclinD1 and cyclinE1, modulation of Bcl-2 family proteins and activation of PARP. In addition, verteporfin exhibited an inhibitory effect on angiogenesis and vasculogenic mimicry via suppressing Ang2, MMP2, VE-cadherin, and α-SMA expression in vitro and in vivo. Mechanism studies demonstrated that verteporfin impaired YAP and TEAD interaction to suppress the expression of targeted genes. Our results provide a foundation for repurposing verteporfin as a promising anti-tumor drug in the treatment of pancreatic cancer by targeting the Hippo pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, CD; Antineoplastic Agents; Apoptosis; Cadherins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin E; DNA-Binding Proteins; G1 Phase Cell Cycle Checkpoints; Human Umbilical Vein Endothelial Cells; Humans; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Nuclear Proteins; Oncogene Proteins; Pancreatic Neoplasms; Phosphoproteins; Poly (ADP-Ribose) Polymerase-1; Porphyrins; Proto-Oncogene Proteins c-bcl-2; TEA Domain Transcription Factors; Transcription Factors; Verteporfin; Vesicular Transport Proteins; Xenograft Model Antitumor Assays; YAP-Signaling Proteins

Delta-24-RGD (DNX-2401) is a conditional replication-competent oncolytic virus engineered to preferentially replicate in and lyse tumor cells with abnormality of p16/RB/E2F pathway. In a phase I clinical trial, Delta-24-RGD has shown favorable safety profile and promising clinical efficacy in brain tumor, which prompted us to evaluate its anticancer activity in pancreatic ductal adenocarcinoma (PDAC), which also has high frequency of homozygous deletion and promoter methylation of CDKN2A encoding the p16 protein. Our results demonstrate that Delta-24-RGD can induce dramatic cytotoxicity in a subset of PDAC cell lines with high cyclin D1 expression. Induction of autophagy and apoptosis by Delta-24-RGD in sensitive PDAC cells was confirmed with LC3B-GFP autophagy reporter and acridine orange staining as well as Western blotting analysis of LC3B-II expression. Notably, we found that Delta-24-RGD induced phosphatidylserine exposure in infected cells independent of cells' sensitivity to Delta-24-RGD, which renders a rationale for combination of Delta-24-RGD viral therapy and phosphatidylserine targeting antibody for PDAC. In a mouse PDAC model derived from a liver metastatic pancreatic cancer cell line, Delta-24-RGD significantly inhibited tumor growth compared with control (

Topics: Animals; Autophagy; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Dependovirus; DNA Methylation; Genetic Therapy; Humans; Liver Neoplasms; Mice; Oncolytic Viruses; Pancreatic Neoplasms; Phosphatidylserines; Promoter Regions, Genetic; Sequence Deletion; Xenograft Model Antitumor Assays

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) belongs to the human carcino-embryonic antigen (CEA) family. Numerous lines of studies have indicated that altered expression of CEACAM6 may have a role in carcinogenesis and development. However, few studies have defined functional roles and mechanisms of action. In the present study, the relationship between clinical and pathological parameters was also analyzed. The relative CEACAM6 protein expression of pancreatic carcinoma was significantly higher than that in non-cancerous tissue. Different clinical stages and lymph node metastasis between groups were significantly different (P<0.05). We used siRNA and forced-expression in multiple cell lines to define the role of CEACAM6 in the regulation of proliferation of pancreatic carcinoma in vitro and in vivo. Knockdown of endogenous CEACAM6 decreased proliferation of BxPC-3 and SW1990 cells. These changes significantly reduced cyclin D1 and CDK4 protein levels. Conversely, overexpression of CEACAM6 in MIA PaCa-2 cells stimulated proliferation and increased cyclin D1 and CDK4 protein levels. Our results confirm that CEACAM6 promoted cell proliferation, and these changes were mediated by cyclin D1/CDK4. These observations contribute to our understanding of the important roles of CEACAM6 in pancreatic carcinoma development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of pancreatic carcinoma.

Topics: Animals; Antigens, CD; Biomarkers, Tumor; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Progression; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Mice; Neoplasm Transplantation; Pancreatic Neoplasms

Recent studies suggest that metformin, which is a commonly used oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, yet the detailed mechanisms by which metformin affects various types of cancers, including pancreatic cancer, remain unknown. The aim of the present study was to evaluate the effects of metformin on human pancreatic cancer cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin. We used the human pancreatic cancer cell lines Panc1, PK1 and PK9 to study the effects of metformin on human pancreatic cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 5 weeks, and the expression of cell cycle-related proteins was determined. In addition, we used miRNA microarray tips to explore the differences in the levels of miRNAs in Panc1 cells and xenograft tumors treated with metformin or without. Metformin inhibited the proliferation of Panc1, PK1 and PK9 cells in vitro. This inhibition was accompanied by a strong decrease in G1 cyclins (particularly in cyclin D1) and retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor (EGFR), particularly the phosphorylation of EGFR at Tyr845, and insulin-like growth factor 1 receptor (IGF-1R) in vitro and in vivo. miRNA expression was markedly altered by the treatment with metformin in vitro and in vivo. Our results revealed that metformin inhibits human pancreatic cancer cell proliferation and tumor growth, possibly by suppressing the cell cycle-related molecules via alteration of miRNAs.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Hypoglycemic Agents; Metformin; Mice; MicroRNAs; Pancreatic Neoplasms; Phosphorylation; Receptor, IGF Type 1; Retinoblastoma Protein; Xenograft Model Antitumor Assays

The transcription factor Krüppel-like factor 8 (KLF8) plays an important role in tumor development and growth, but its role in pancreatic cancer (PC) is not clear.. KLF8 expression in human PC cell lines and tumor tissues was measured by quantitative real-time polymerase chain reaction and Western blot analyses. The effects of lentivirus mediated knockdown of KLF8 on proliferation and growth in Panc-1 pancreatic cancer cells were examined.. KLF8 was overexpressed in 5 pancreatic cancer cell lines and in samples from patients with PC. In Panc-1 cells, KLF8 knockdown inhibited cell proliferation, tumorigenicity, and induced G2/M phase arrest. KLF8 knockdown suppressed PC tumor growth in nude mice model. Western blot analysis showed that KLF8 knockdown in Panc-1 cells down-regulated the expression of CDK1/CDC2, cyclin B1, and cyclin D1 and up-regulated the expression of p21, and p27.. Overexpression of KLF8 may contribute to the progression of pancreatic cancer, and downregulation of KLF8 expression by lentivirus-delivered shRNA is a novel therapeutic approach for PC.

Topics: Animals; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Real-Time Polymerase Chain Reaction; Repressor Proteins; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous

Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degron(ODC) (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-Kras(G12D) model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation.

Topics: Animals; Carcinogenesis; Ceruletide; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; eIF-2 Kinase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Integrases; Mice; Mice, Transgenic; NF-kappa B; Pancreas; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins p21(ras); Signal Transduction; Trans-Activators

Disheveled-axin (DIX) domain containing 1 (DIXDC1), a protein containing a coiled-coil domain and a DIX domain, is involved in the progression of multiple cancers. However, the role of DIXDC1 in human pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, we investigated the role and prognostic value of DIXDC1 in the development of human PDAC. Western blot analysis revealed that DIXDC1 was highly expressed in PDAC tissues and cell lines. Immunohistochemistry on 165 paraffin-embedded sections showed that high expression of DIXDC1 was significantly correlated with tumor size (P = .002), histological differentiation (P = .001), tumor node metastasis (TNM) stage (P = .001), and the proliferation marker Ki-67 (P = .000). Importantly, Kaplan-Meier analysis revealed that high expression of DIXDC1 was obviously correlated with worsened overall survival (P < .001). In vitro, using serum starvation-refeeding experiments, our results suggested that DIXDC1 was up-regulated in proliferating PDAC cells, together with the percentage of cells at the S phase, and was correlated with the expression of cyclin D1. In addition, depletion of DIXDC1 decreased PCNA and cyclin D1 levels. Accordingly, CCK-8, colony formation, and flow cytometry analyses revealed that knocking down DIXDC1 induced growth impairment and G1/S cell cycle arrest in PDAC cells, while overexpression of DIXDC1 led to accelerated cell proliferation and cell cycle progression. On the basis of these results, we propose that DIXDC1 could play an important role in the tumorigenesis of PDAC and serve as a potential therapeutical target to prevent PDAC progression.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Microfilament Proteins; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; RNA Interference; Signal Transduction; Time Factors; Transfection; Tumor Burden; Up-Regulation

Transcription factor SOX18 has been proved to play a significant role in carcinogenesis. However, no investigation was performed about the expression of SOX18 in pancreatic ductal adenocarcinoma (PDAC). In our work, we found that the PDAC tissues had higher level of SOX18 mRNA and protein expression than matched non-tumor pancreatic tissues and high level of SOX18 protein indicated poor prognosis for PDAC patients. After knockdown of SOX18 gene in PANC-1 and SW1990 cell lines, which showed higher expression level of SOX18 among five PDAC cell lines, the abilities of proliferation, migration and invasion were inhibited and the tumor growth was suppressed in vivo. In addition, the flow cytometry results indicated that down-regulation of SOX18 induced G1/S phase arrest. Furthermore, we found that the expression of cyclin D1, c-myc and MMP-7, three tumorigenesis promoters, was inhabited with downregulation of SOX18. In conclusion, our study reveals that SOX18 plays a significant role in promoting the growth of PDAC, and might serve as a promising target for PDAC therapy.

Topics: Aged; Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 7; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Transplantation; Pancreatic Neoplasms; Prognosis; Proto-Oncogene Proteins c-myc; RNA, Messenger; SOXF Transcription Factors; Transfection

CXCR4 and glycogen synthase kinase-3β (GSK3β) promote proliferation and invasion of pancreatic cancer. Inhibition of CXCR4 suppresses GSK3β expression. However, the molecular mechanism by which CXCR4 contributes to human pancreatic cancer metastasis is not completely understood. In this study, therefore, we analyzed the effect of CXCR4 on GSK3β expression and its molecular mechanism.. PANC-1 and SW-1990 cells were used in this study. PANC-1 and SW-1990 cell lines which stably expressed upregulated or downregulated CXCR4 were used for further study. Western blotting was employed to detected the expression of CXCR4, GSK3β and MMP-2. Cell invasion assay was used to detect the effect of the Akt pathway on CXCR4-induced GSK3β expression.. Overexpression of CXCR4 promoted GSK3β expression and silencing of CXCR4 suppressed GSK3β expression. Overexpression of CXCR4 activated cyclin D1 and p-Akt expression, but inhibited p21 expression. Silencing of CXCR4 had the reverse effect. CXCR4 promoted GSK3β expression and PANC-1 invasion by Akt signaling. CXCR4 upregulated GSK3β expression, at least in part, at the level of transcription.. CXCR4 promotes GSK3β expression via the Akt cell signaling pathway in pancreatic cancer cells.

Topics: Adenocarcinoma; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Neoplasm Invasiveness; Pancreatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, CXCR4; Signal Transduction

CRKL is an adapter protein which is overexpressed in many malignant tumors and plays crucial roles in tumor progression. However, expression pattern and biological roles of CRKL in pancreatic cancer have not been examined. In the present study, we found that CRKL expression in pancreatic cancer specimens was higher than that in normal pancreatic tissues. Colony formation assay and Matrigel invasion assay showed that the overexpression of CRKL in Bxpc3 and Capan2 cell lines with low endogenous expression increased cell proliferation and invasion. Flow cytometry showed that CRKL promoted cell proliferation by facilitating cell cycle. Further analysis of cell cycle- and invasion-related molecules showed that CRKL upregulated cyclin D1, cyclin A, matrix metalloproteinase 2 (MMP2) expression, and phosphorylated extracellular signal (ERK)-regulated kinase. In conclusion, our study demonstrated that CRKL was overexpressed in human pancreatic cancers and contributed to pancreatic cancer cell proliferation and invasion through ERK signaling.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Nuclear Proteins; Pancreatic Neoplasms; Signal Transduction

Pancreatic cancer, the 4th leading cause of cancer death in the US, is highly resistant to all current chemotherapies, and its growth is facilitated by chronic inflammation. An important mediator of inflammation is the nuclear factor kappa B (NFκB), a transcription factor that regulates over 500 genes including the regulation of anti-apoptotic proteins, cell cycle progression and cytokine production. NFκB is constitutively activated in pancreatic cancer cells contributing to their resistance to apoptosis and high metastatic potential. Although many small molecules that inhibit NFκB have been identified, none are currently used in the clinic, perhaps due to their lack of specificity. To identify novel inhibitors of NFκB, the HBOI library of enriched fractions from marine organisms was screened using a reporter cell line that produces luciferin under the transcriptional control of NFκB. Fractions from the sponge Amphibleptula were active in this screen and contained the antifungal cyclic peptide microsclerodermin A. Microsclerodermin A is shown here to inhibit NFκB transcriptional activity in a reporter cell line, to reduce levels of phosphorylated (active) NFκB in the AsPC-1 cell line, to have an IC50 for cytotoxicity in the low micromolar range against the AsPC-1, BxPC-3, MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, and to induce significant apoptosis in the AsPC-1, BxPC-3 and the PANC-1 cell lines. Treatment of AsPC-1 cells with microsclerodermin A also resulted in an increase in IL-8 production without apparent induction of angiogenic factors and there is the possibility that inhibition of NFκB by microsclerodermin A is mediated by the glycogen synthase kinase 3β pathway.

Topics: Antineoplastic Agents; Apoptosis; Biological Products; Caspases; Cell Line, Tumor; Cell Survival; Cyclin D1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Interleukin-8; JNK Mitogen-Activated Protein Kinases; NF-kappa B; Pancreatic Neoplasms; Peptides, Cyclic; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos

Cyclin D1 is an important regulator protein for the G1-S cell cycle phase transition. The aim of this trial was to evaluate the impact of the CCND1 polymorphism G870A and corresponding protein expression and CCND1 amplification on the survival of the patients.. 425 patients with ductal pancreatic adenocarcinoma who underwent resection were included after histopathological confirmation. DNA was analyzed for Cyclin D1 polymorphisms, immunhistochemical examination and fluorescence in situ hybridization analysis of the tumor were performed.. Overall, the mean survival was 22.9 months (20.5-25.3). The survival in patients with Cyclin D1 G870A polymorphism Adenine/Adenine was 15.1 months (95% CI 11.3-18.9), 21.5 months (17.4-25.6) for Adenine/Guanine, and 29.4 months (95% CI 23.8-35.0) for Guanine/Guanine (P = 0.003). A shorter survival was associated with strong/moderate protein expression in immunohistochemistry (IHC) compared to weak/no expression (P = 0.028). Additionally, a significant coherency between unfavourable polymorphism (AA/AG) and increased protein expression was detected (P = 0.005).. A strong impact on survival of Cyclin D1 G870A polymorphism and the detected corresponding protein expression was found. The biological mechanism of CCND1 in carcinogenesis has not been fully examined; but at present Cyclin D1 seems to be an interesting biomarker for the prognosis of ductal adenocarcinoma.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cyclin D1; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Pancreatic Neoplasms; Polymorphism, Genetic; Prognosis; Prospective Studies; Survival Rate

Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Aspirin; Bcl-2-Like Protein 11; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Female; Humans; Membrane Proteins; Metformin; Mice; Mice, SCID; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Transplantation; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; TOR Serine-Threonine Kinases; Wound Healing

Previous population investigations have suggested that the application of curcumin may be associated with decreased incidence and improved prognosis in certain types of cancer. Forkhead box O1 (FOXO1) has been implicated in the regulation of several biological processes, including stress resistance, metabolism, DNA repair, cell cycle and apoptosis. The aims of the present study were to investigate the effects and molecular mechanisms of curcumin on the induction of anti‑proliferation, cell cycle arrest and apoptosis, by FOXO1, in pancreatic cancer cells. The MTT assay and ELISA‑Brdu assay were used to assess cell proliferation. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to detect the expression of PCNA, Ki‑67, B‑cell lymphoma‑2 (Bcl‑2), B‑cell‑associated X protein (Bax), cyclin D1, p21, p27 and FOXO1. Cell apoptosis was detected using a Cell Death ELISA detection kit. A Caspase‑3/9 Fluorescent Assay kit was used to detect caspase activity. The findings revealed that curcumin significantly decreased cell proliferation, which was associated with increased expression of the p21/CIP1 and p27/KIP1 cyclin‑dependent kinase inhibitors, and inhibited expression of cyclin D1. In addition, curcumin induced apoptosis by decreasing the Bcl‑2/Bax protein ratio and increasing caspase‑9/3 activation in the pancreatic cancer cells. Using siRNA against FOXO1, and Akt inhibitor and activator, the present study confirmed that curcumin induced the expression of FOXO1 by inhibition of phosphoinositide 3‑kinase/Akt signaling, leading to cell cycle arrest and apoptosis. In conclusion, these findings offer support for a mechanism that may underlie the anti‑neoplastic effects of curcumin and justify further investigation to examine the potential roles for activators of FOXO1 in the prevention and treatment of pancreatic cancer.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

B7-H1, a member of the B7 family of proteins, is hypothesised to play an important role in the immune escape of tumours through its binding to the PD-1 receptor on activated T and B cells. By inducing T lymphocyte apoptosis, tumour cells can suppress an effective antitumour immune response. Although the immunosuppressive effect of B7-H1 has been studied in many tumours, its other biological functions remain unclear. We previously demonstrated a high expression level of this molecule in pancreatic cancer patient samples and its antiapoptotic effect in pancreatic ductal adenocarcinoma (PDA) cells. The aim of the present study was to investigate the possible role of B7-H1 in the proliferation of PDA cells. Functional studies were performed using pancreatic cell lines that were genetically engineered to express high or low levels of B7-H1, and we found that the overexpression of B7-H1 through plasmid transfection in PDA cells promoted cell proliferation. Conversely, the short-hairpin RNA (shRNA) knockdown of B7-H1 inhibited PDA cell proliferation. Further analyses of the cell cycle and cell cycle-related molecules confirmed this result. Taken together, our results indicate that the upregulation of B7-H1 in pancreatic cancer cells promotes proliferation and accelerates carcinogenesis; these data, therefore, provide insights into the effects of B7-H1 overexpression on pancreatic tumour cells.

Topics: B7-H1 Antigen; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; G2 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 4; Pancreatic Neoplasms; Up-Regulation

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.

Topics: Animals; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Guanine Nucleotide Exchange Factors; Heterografts; Humans; Lung Neoplasms; Mice; Mice, SCID; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Protein Isoforms; rho GTP-Binding Proteins

The objective of this study was to investigate the expression and clinical relevance of interleukin 22 (IL-22) and IL-22-producing CD4 T cells (IL-22CD4 T cells) in pancreatic cancer (PC) tissues.. Interleukin 22 protein levels in PC tissues were measured by Western blot analysis and immunohistochemistry. The frequencies of IL-22CD4 T cells in tumors and peripheral blood from PC patients and control subjects were analyzed by flow cytometry. The association between IL-22 and phosphorylation of STAT-3 was investigated in in vitro model.. Interleukin 22 protein was more highly expressed in PC tissues than in peritumoral and normal pancreatic tissues. The frequencies of all IL-22CD4 T cells and T helper 22 (TH22) cells (IL-22IFN-γIL-17CD4) were significantly higher in PC tissues than in the peripheral blood of PC patients and control subjects. It was observed that up-regulation pSTAT-3 and its downstream genes such as Bcl-2 and cyclin D1 in vitro. Finally, we found that increased intratumoral IL-22 expression and frequencies of TH22 and IL-22CD4 T cells were positively correlated with PC tumor-node-metastasis staging.. Increased intratumoral IL-22 levels, IL-22CD4 T cells, and TH22 cells are correlated with PC tumor-node-metastasis staging, suggesting that IL-22 and IL-22CD4 T cells may be related to tumor progression and are potential therapeutic targets in patients with PC.

Topics: Blotting, Western; CD4-Positive T-Lymphocytes; Cells, Cultured; Cyclin D1; Disease Progression; Female; Flow Cytometry; Humans; Immunohistochemistry; Interleukin-22; Interleukins; Male; Middle Aged; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; T-Lymphocytes, Helper-Inducer; Up-Regulation

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.

Topics: Animals; Blotting, Western; Carcinoma, Pancreatic Ductal; Cell Culture Techniques; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Oncogenes; Pancreatic Ducts; Pancreatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Telomerase; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

The prognostic significance of cyclin D1, p16(INK) (4A) and p27(Kip1) expression has been documented in several human malignancies; however, their prognostic potential in pancreatic adenocarcinoma is still unclear. This study aimed to assess the correlation of the aforementioned molecules with clinicopathological parameters and prognosis. Sixty patients with pancreatic ductal adenocarcinoma underwent surgical resection at a single institution; immunohistochemical staining of the studied markers was quantified by Ιmage analysis system. Cyclin D1 overexpression was positively associated with grade, neural infiltration and vascular invasion, whereas p27 positively correlated with age. Higher cyclin D1 expression indicated poorer survival (adjusted HR = 9.75, 95%CI: 1.48-64.31, p = 0.018, increment: one unit in H-score), whereas a marginal trend toward an association between p16 positivity and improved survival was observed (adjusted HR = 0.58, 95%CI: 0.32-1.05, p = 0.072 regarding positive vs negative cases). No significant association with overall survival was noted regarding p27. In conclusion, cyclin D1 overexpression and possibly p16 loss of expression in pancreatic adenocarcinoma seem to be adverse prognostic factors, whereas p27 expression did not seem to possess such prognostic properties. Further validation of the present findings in studies encompassing larger samples seems to be needed.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Prognosis; Proportional Hazards Models; Retrospective Studies

Pancreatic adenocarcinoma up-regulated factor (PAUF) is expressed in pancreatic ductal adenocarcinoma (PDAC) and plays an important role in tumor progression and metastasis. Here we evaluate the anti-tumor efficacy of a human monoclonal antibody against PAUF, PMAb83, to provide a therapeutic intervention to treat the disease. PMAb83 reduced tumor growth and distant metastasis in orthotopically xenografted mice of human PDAC cells. PMAb83 treatment retarded proliferation along with weakened aggressiveness traits of the carcinoma cells. AKT/β-catenin signaling played a role in the carcinoma cell proliferation and the treated xenograft tumors exhibited reduced levels of β-catenin and cyclin D1. Moreover PMAb83 abrogated the PAUF-induced angiogenic responses of endothelial cells, reducing the density of CD31(+) vessels in the treated tumors. In combination with gemcitabine, PMAb83 conferred enhanced survival of xenografted mice by about twofold compared to gemcitabine alone. Taken together, our findings show that PMAb83 treatment decreases the aggressiveness of carcinoma cells and suppresses tumor vascularization, which culminates in mitigated tumor growth and metastasis with improved survival in PDAC mouse models.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antineoplastic Agents; beta Catenin; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Combined Modality Therapy; Cyclin D1; Deoxycytidine; Disease Progression; Endothelial Cells; Female; Gemcitabine; Humans; Intercellular Signaling Peptides and Proteins; Lectins; Mice; Mice, Nude; Neovascularization, Pathologic; Pancreatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Xenograft Model Antitumor Assays

Pancreatic ductal adenocarcinoma has a poor prognosis due to late diagnosis and a lack of effective therapeutic options. Thus, it is important to better understand its molecular mechanisms and to develop more effective treatments for the disease. The ternary complex factor Net, which exerts its strong inhibitory function on transcription of proto-oncogene gene c-fos by forming ternary complexes with a second transcription factor, has been suspected of being involved in pancreatic cancer and other tumors biology. In this study, we found that the majority of pancreatic ductal adenocarcinoma tissues and cell lines had weak or no expression of Net, whereas significantly high level of Net expression occurred in paired adjacent normal tissues we studied. Furthermore, using in vitro and in vivo model systems, we found that overexpression of Net inhibited cell growth and survival and induced cell apoptosis in human pancreatic ductal adenocarcinoma cell PL45; the mechanisms by which Net inhibited the cell cycle progression were mainly through P21-Cyclin D1/CDK4 Pathway. Our data thus suggested that Net might play an important role in pancreatic carcinogenesis, possibly by acting as a tumor suppressor gene.

Topics: Adult; Aged; Animals; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Middle Aged; Norepinephrine Plasma Membrane Transport Proteins; Pancreatic Neoplasms; Proto-Oncogene Mas; Signal Transduction

Six1 is one of the transcription factors that act as master regulators of development and are frequently dysregulated in cancers. However, the role of Six1 in pancreatic cancer is not clear. Here we show that the relative expression of Six1 mRNA is increased in pancreatic cancer and correlated with advanced tumor stage. In vitro functional assays demonstrate that forced overexpression of Six1 significantly enhances the growth rate and proliferation ability of pancreatic cancer cells. Knockdown of endogenous Six1 decreases the proliferation of these cells dramatically. Furthermore, Six1 promotes the growth of pancreatic cancer cells in a xenograft assay. We also show that the gene encoding cyclin D1 is a direct transcriptional target of Six1 in pancreatic cancer cells. Overexpression of Six1 upregulates cyclin D1 mRNA and protein, and significantly enhances the activity of the cyclin D1 promoter in PANC-1 cells. We demonstrate that Six1 promotes cell cycle progression and proliferation by upregulation of cyclin D1. These data suggest that Six1 is overexpressed in pancreatic cancer and may contribute to the increased cell proliferation through upregulation of cyclin D1.

Topics: Analysis of Variance; Animals; Blotting, Western; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; DNA Primers; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Mice; Mice, Inbred BALB C; Microarray Analysis; Pancreatic Neoplasms; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Tumor Stem Cell Assay

Conventional classifications of gastroenteropancreatic neuroendocrine tumours (GEP- NETs) are rather unsatisfactory because of the variation in survival within each subgroup. Molecular markers are being found able to predict patient outcome in more and more tumours. The aim of this study was to characterize the expression of the proteins cyclin D1, cyclin E and P53 in GEP- NETs and assess any prognostic impact. Tumor specimens from 68 patients with a complete follow-up were studied immunohistochemically for cyclin D1, cyclin E and P53 expression. High cyclin D1 and cyclin E immunostaining (≥ 5% positive nuclei) was found in 48 (71%) and 24 (35%) cases, and high P53 staining (≥ 10% positive nuclei) in 33 (49%) . High expression of P53 was more common in gastric neuroendocrine tumors and related to malignant behavior, being associate with a worse prognosis on univariate analysis (RR=1.9, 95%CI=1.1-3.2). High expression of cyclin E was significantly associated with shorter survival in the univariate analysis (RR=2.0, 95%CI=1.2-3.6) and multivariate analysis (RR=2.1, 95%CI=1.1-4.0). We found no significant correlation between the expression of cyclin D1 and any clinicopathological variables. Our study indicated a prognostic relevance for cyclin E and P53 immunoreactivity. Cyclin E may be an independent prognostic factor from the 2010 WHO Classification which should be evaluated in further studies.

Topics: Aged; Colorectal Neoplasms; Cyclin D1; Cyclin E; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Neuroendocrine Tumors; Pancreatic Neoplasms; Proportional Hazards Models; Retrospective Studies; Stomach Neoplasms; Tumor Suppressor Protein p53

Topics: Adaptor Proteins, Vesicular Transport; Animals; Carcinoma, Pancreatic Ductal; Cell Cycle; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Mice; Myeloid Differentiation Factor 88; Pancreatic Neoplasms; Signal Transduction; Stromal Cells; Toll-Like Receptor 4; Toll-Like Receptor 7

Cyclin D1 is important for pancreatic cancer growth. Our aim was to determine the effects of cyclin D1 inhibition on the growth of established pancreatic tumors.. PANC-1 cells harboring cyclin D1 antisense cDNA in a tetracycline-inducible vector system were prepared. The effects of cyclin D1 inhibition after tumor development were characterized in a mouse model.. In vitro removal of tetracycline induced cyclin D1 antisense cDNA expression and inhibited cyclin D1 expression and cyclin D1-associated kinase activity as well as anchorage-dependent and -independent growth. After establishment of xenograft tumors in the presence of tetracycline (2 mg/mL) in the drinking water, animals were assigned to either control (tetracycline remained in the drinking water) or to the group without tetracycline for which tetracycline was removed from the drinking water. Tumor growth was significantly inhibited after removal of tetracycline. Microscopic analysis revealed that the area of central necrosis was significantly increased in the group without tetracycline paralleled by a reduction of the vital peripheral area of proliferating cells.. Our results confirmed that cyclin D1 plays an important role in the growth of pancreatic cancer cells and may be an attractive molecular target for the treatment of human pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA, Antisense; Female; Gene Expression Regulation; Genes, Reporter; Genetic Therapy; Humans; Mice; Mice, Nude; Necrosis; Pancreatic Neoplasms; Tetracycline; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Therapeutic strategies based on modulation of microRNA activity possess much promise in cancer therapy, but the in vivo delivery of microRNA to target sites and its penetration into tumor tissues remain great challenge. In this work, miR-34a-delivering therapeutic nanocomplexes with a tumor-targeting and -penetrating bifunctional CC9 peptide were proposed for efficient treatment of pancreatic cancers. In vitro study indicated that the nanoparticle-based miR-34a delivery systems could effectively facilitate cellular uptake and greatly up-regulate the mRNA level of miR-34a in PANC-1 cell lines. The up-regulation of miR-34a remarkably induced cell cycle arrest and apoptosis, suppressed the tumor cell migration and inhibited the target gene expressions such as E2F3, Bcl-2, c-myc and cyclin D1. More importantly, the in vivo systemic administration of the developed targeting miR-34a delivery systems in a pancreatic cancer model significantly inhibited tumor growth and induced cancer cell apoptosis. Such bifunctional peptide-conjugated miRNA-delivering nanocomplexes should have great potential applications in cancer therapy.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; E2F3 Transcription Factor; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Humans; Mice; Mice, Inbred BALB C; MicroRNAs; Nanoparticles; Nanotechnology; Pancreatic Neoplasms; Peptides; Proto-Oncogene Proteins c-myc; Up-Regulation; Xenograft Model Antitumor Assays

Up-regulation of phosphatidylinositol-3-kinase (PI3K)-AKT signaling facilitates tumor cell growth and inhibits cell demise. The AKT-pathway also plays an important role in cytostatic therapy resistance and response to hypoxia and angiogenesis. Using real-time cell proliferation assay we examined the potency of triciribine in three distinct neuroendocrine gastrointestinal tumor cell lines. Also we investigated triciribine's induction of apoptosis and effects on a broad range of cancer-associated gene products. Furthermore, we characterized the role of PTEN as a possible predictor of sensitivity to triciribine in GEP-NETs. We also looked for additive anti-neoplastic effects of triciribine when combined with conventional cytostatic drugs or other targeted drugs, affecting different molecules of the PI3K-AKT-pathway and we assessed the potency of triciribine to inhibit tumor growth in vivo, by using the chick chorioallantoic membrane assay. Treatment of insulinoma (CM) or gut neuroendocrine tumor cells (STC-1) with triciribine significantly reduced tumor cell growth by 59% and 65%, respectively. By contrast, the highly expressing PTEN carcinoid cell line BON did not respond, even at higher doses. Combinations of triciribine with classic cytostatic drugs as well as drugs targeting other molecules of the PI3K-AKT-pathway led to synergistic anti-proliferative effects. Additional in vivo-evaluations confirmed the anti-neoplastic potency of triciribine. Thus, our data show that inhibition the AKT-pathway potently reduces the growth of GEP-NET cells alone or in combination therapies. AKT inhibition may provide a rationale for future evaluations.

Topics: Aging; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gastrointestinal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Insulinoma; Neuroendocrine Tumors; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribonucleosides; Signal Transduction

Pancreatic cancer (PC) is the most aggressive malignant disease, ranks as the fourth most leading cause of cancer-related death among men and women in the United States. We present here that plumbagin (PL), a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L, inhibits the growth of PC cells both in vitro and in vivo model systems. PL treatment induces apoptosis and inhibits cell viability of PC cells (PANC1, BxPC3 and ASPC1). In addition, i.p. administration of PL (2 mg/kg body weight, 5 days a week) in severe combined immunodeficiency (SCID) mice beginning 3 days after ectopic implantation of PANC1 cells resulted in a significant (P < 0.01) inhibition of both tumor weight and volume. PL treatment inhibited (1) constitutive expression of epidermal growth factor receptor (EGFR), pStat3Tyr705 and pStat3Ser727, (2) DNA binding of Stat3 and (3) physical interaction of EGFR with Stat3, in both cultured PANC1 cells and their xenograft tumors. PL treatment also inhibited phosphorylation and DNA-binding activity of NF-κB in both cultured PC cells (PANC1 and ASPC1) and in PANC1 cells xenograft tumors. Downstream target genes (cyclin D1, MMP9 and Survivin) of Stat3 and NF-κB were similarly inhibited. These results suggest that PL may be used as a novel therapeutic agent against human PC. Published 2012 Wiley-Liss, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; ErbB Receptors; Humans; Inhibitor of Apoptosis Proteins; Male; Matrix Metalloproteinase Inhibitors; Mice; Mice, SCID; Naphthoquinones; NF-kappa B; Pancreatic Neoplasms; Phosphorylation; Plant Extracts; Signal Transduction; STAT3 Transcription Factor; Survivin

Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of β-catenin, we postulated that β-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target β-catenin in a colon cancer model, suppresses β-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of β-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced β-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of β-catenin. Treatment with MG132, a proteasomal inhibitor, restored β-catenin protein levels, suggesting that SIRT1-mediated degradation of β-catenin requires proteasomal activity. It was reported that inhibition of GSK-3β or Siah-1 stabilizes β-catenin in colon cancer cells, but suppression of GSK-3β or Siah-1 using siRNA in the presence of resveratrol instead diminished β-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3β and Siah-1 are not involved in SIRT1-mediated degradation of β-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of β-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of β-catenin.

Topics: beta Catenin; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Lectins; Leupeptins; Oncogenes; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Sirtuin 1

In mice, genetic changes that inactivate the retinoblastoma tumor suppressor pathway often result in pancreatic neuroendocrine tumors (Pan-NETs). Conversely, in humans with this disease, mutations in genes of the retinoblastoma pathway have rarely been detected, even in genome-wide sequencing studies. In this study, we took a closer look at the role of the retinoblastoma pathway in human Pan-NETs.. Pan-NET tumors from 92 patients were subjected to immunohistochemical staining for markers of the retinoblastoma pathway. To search for amplifications of retinoblastoma pathway genes, genomic DNAs from 26 tumors were subjected to copy number analysis. Finally, a small-molecule activator of the retinoblastoma pathway was tested for effects on the growth of two Pan-NET cell lines.. A majority of tumors expressed high amounts of Cdk4 or its partner protein cyclin D1. High amounts of phosphorylated Rb1 were present in tumors that expressed high levels of Cdk4 or cyclin D1. The copy numbers of Cdk4 or the analogous kinase gene Cdk6 were increased in 19% of the tumors. Growth of the human Pan-NET cell line QGP1 was inhibited in a xenograft mouse model by the Cdk4/6 inhibitor, PD 0332991, which reactivates the retinoblastoma pathway.. Inactivation of the retinoblastoma pathway was indicated for most Pan-NETs. Gene amplification and overexpression of Cdk4 and Cdk6 suggests that patients with Pan-NETs may respond strongly to Cdk4/6 inhibitors that are entering clinical trials.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; DNA Copy Number Variations; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kaplan-Meier Estimate; Male; Metabolic Networks and Pathways; Mice; Middle Aged; Mutation; Neoplasm Staging; Neuroendocrine Tumors; Pancreatic Neoplasms; Piperazines; Pyridines; Retinoblastoma Protein; Transplantation, Heterologous

The factors preventing the translation of preclinical findings supporting the clinical development mTOR-targeted therapy in pancreatic cancer therapy remain undetermined. Stromal cell.derived factor 1α (SDF-1α)-CXCR4 signaling was examined as a representative microenvironmental factor able to promote mTOR-targeted therapy resistance in pancreatic cancer.. Primary pancreas explant xenografts and in vitro experiments were used to perform pharmacodynamic analyses of SDF-1α-CXCR4 regulation of the mTOR pathway. Combinatorial effects of CXCR4, EGFR, and mTOR pharmacologic inhibition were evaluated in temsirolimus-resistant and -sensitive xenografts. Intratumoral gene and protein expressions of mTOR pathway effectors cyclin D1, c-Myc, and VEGF were evaluated.. Baseline intratumoral SDF-1α gene expression correlated with temsirolimus resistance in explant models. SDF-1α stimulation of pancreatic cells resulted in CXCR4-mediated PI3-kinase-dependent S6-RP phosphorylation (pS6-RP) on exposure to temsirolimus. Combinatorial therapy with AMD3465 (CXCR4 small-molecule inhibitor) and temsirolimus resulted in effective tumor growth inhibition to overcome temsirolimus resistance. In contrast, SDF-1α exposure induced a temsirolimus-resistant phenotype in temsirolimus-sensitive explants. AMD3465 inhibited CXCR4-mediated intratumoral S6-RP phosphorylation and cyclin D and c-myc gene expression. Next, CXCR4 promoted intratumoral EGFR expression in association with temsirolimus resistance. Treatment with AMD3465, temsirolimus- and erlotinib-mediated tumor growth inhibition to overcome temsirolimus resistance in the explant model. Lastly, SDF-1α-CXCR4 signaling increased intratumoral VEGF gene and protein expression.. SDF-1α-CXCR4 signaling represents a microenvironmental factor that can maintain mTOR pathway fidelity to promote resistance to mTOR-targeted therapy in pancreatic cancer by a variety of mechanisms such as recruitment of EGFR signaling and angiogenesis.

Topics: Animals; Cell Line, Tumor; Chemokine CXCL12; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Pyridines; Quinazolines; Receptors, CXCR; Receptors, CXCR4; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterologous; Tumor Microenvironment; Vascular Endothelial Growth Factor A

Nitric oxide-releasing aspirin (NO-aspirin) represents a novel class of promising chemopreventive agents. Unlike conventional nonsteroidal anti-inflammatory drugs, NO-aspirin seems to be free of adverse effects while retaining the beneficial activities of its parent compound. The effect of NO-aspirin on pancreatic carcinogenesis was investigated by assessing the development of precursor pancreatic lesions and adenocarcinomas in Kras(G12D/+) transgenic mice that recapitulate human pancreatic cancer progression. Six-week-old male p48(Cre/+)-LSL-Kras(G12D/+) transgenic mice (20 per group) were fed diets containing 0, 1000, or 2000 ppm NO-aspirin. The development of pancreatic tumors was monitored by positron emission tomography imaging. All mice were killed at the age of 41 weeks and assessed for pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) and for molecular changes in the tumors. Our results reveal that NO-aspirin at 1000 and 2000 ppm significantly suppressed pancreatic tumor weights, PDAC incidence, and carcinoma in situ (PanIN-3 lesions). The degree of inhibition of PanIN-3 and carcinoma was more pronounced with NO-aspirin at 1000 ppm (58.8% and 48%, respectively) than with 2000 ppm (47% and 20%, respectively). NO-aspirin at 1000 ppm significantly inhibited the spread of carcinoma in the pancreas (∼97%; P < .0001). Decreased expression of cyclooxygenase (COX; with ∼42% inhibition of total COX activity), inducible nitric oxide synthase, proliferating cell nuclear antigen, Bcl-2, cyclin D1, and β-catenin was observed, with induction of p21, p38, and p53 in the pancreas of NO-aspirin-treated mice. These results suggest that low-dose NO-aspirin possesses inhibitory activity against pancreatic carcinogenesis by modulating multiple molecular targets.

Topics: Animals; Anticarcinogenic Agents; Aspirin; beta Catenin; Body Weight; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Progression; Humans; Integrases; Male; Mice; Mice, Transgenic; Nitric Oxide Synthase Type II; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Tumor Suppressor Protein p53

To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms.. Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC)] and pancreatic cancer cells (Capan-1 and MIA PaCa-2) were treated with the ceramide analogs (C2, AL6, C6, and C8), at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1) and thrombospondin-1 (TSP-1) mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD) gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice.. Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine.. Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1.

Topics: Administration, Metronomic; Animals; Apoptosis; Caveolin 1; Cell Line, Tumor; Cell Movement; Cell Proliferation; Ceramides; Cyclin D1; Endothelial Cells; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neovascularization, Pathologic; Pancreatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Thrombospondin 1; Tumor Burden; Xenograft Model Antitumor Assays

Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in breast and pancreatic cancer. Inhibiting constitutive STAT3 signaling represents a promising molecular target for therapeutic approach. Using structure-based design, we developed a non-peptide cell-permeable, small molecule, termed as XZH-5, which targeted STAT3 phosphorylation. XZH-5 was found to inhibit STAT3 phosphorylation (Tyr705) and induce apoptosis in human breast and pancreatic cancer cell lines expressing elevated levels of phosphorylated STAT3. XZH-5 could also inhibit interleukin-6-induced STAT3 phosphorylation in cancer cell lines expressing low phosphorylated STAT3. Inhibition of STAT3 signaling by XZH-5 was confirmed by the down-regulation of downstream targets of STAT3, such as Cyclin D1, Bcl-2, and Survivin at mRNA level. In addition, XZH-5 inhibited colony formation, cell migration, and enhanced the cytotoxicity of chemotherapeutic drugs when combined with Doxorubicin or Gemcitabine. Our results indicate that XZH-5 may be a potential therapeutic agent for breast and pancreatic cancers with constitutive STAT3 signaling.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin D1; Female; HeLa Cells; Histidine; Humans; Inhibitor of Apoptosis Proteins; Interleukin-6; Mice; Mice, SCID; Pancreatic Neoplasms; Phenylurea Compounds; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT1 Transcription Factor; STAT3 Transcription Factor; Survivin

Preclinical studies suggest that diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be beneficial for prevention of pancreatic cancer. Nutritional intervention studies are often complex, and there is no clear evidence, without potential confounding factors, on whether conversion of n-6 PUFAs to n-3 PUFAs in pancreatic tissues would provide protection. Experiments were designed using n-3 fatty acid desaturase (Fat-1) transgenic mice, which can convert n-6 PUFA to n-3 FAs endogenously, to determine the impact of n-3 PUFAs on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC). Six-week-old female p48(Cre/+)-LSL-Kras(G12D/+) and compound Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice were fed (AIN-76A) diets containing 10% safflower oil for 35 weeks. Pancreata were evaluated histopathologically for PanINs and PDAC. Results showed a dramatic reduction in incidence of PDAC (84%; P < .02) in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. Importantly, significant reductions of pancreatic ducts with carcinoma (90%; P < .0001) and PanIN 3 (~50%; P < .001) lesions were observed in the compound transgenic mice. The levels of n-3 PUFA were much higher (>85%; P < .05-0.01) in pancreas of compound transgenic mice than in those of p48(Cre/+)-LSL-Kras(G12D/+) mice. Molecular analysis of the pancreas showed a significant down-regulation of proliferating cell nuclear antigen, cyclooxygenase-2, 5-lipoxygenase (5-LOX), 5-LOX-activating protein, Bcl-2, and cyclin D1 expression levels in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. These data highlight the promise of dietary n-3 FAs for chemoprevention of pancreatic cancer in high-risk individuals.

Topics: 5-Lipoxygenase-Activating Proteins; Adenocarcinoma; Animals; Apoptosis; Arachidonate 5-Lipoxygenase; Carcinoma in Situ; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Disease Progression; Down-Regulation; Fatty Acid Desaturases; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Female; Mice; Mice, Transgenic; Pancreatic Ducts; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2

The neuronal pentraxin II gene (NPTX2) is expressed in numerous tissues, such as the pancreas and the liver. While its activity in the brain is known to be regulated by neuronal activity, its function in the pancreas is unclear. In this study, we investigated the impact of NPTX2 on the proliferation, migration, invasion, apoptosis, and cell cycle of the pancreatic cancer cells. The expression levels of NPTX2 and their relation to the methylation level of the NPTX2 gene promoter in five pancreatic cancer cell lines were observed. The lower expression of NPTX2 in the cells was restored after the treatment of DNA methyltransferase inhibitor (5-aza-2'-deoxycytidine). Additionally, a full-length NPTX2 cDNA was transfected into pancreatic cancer cells (PANC-1) and we obtained the stably transfected cells (PANC-1-NPTX2). The ectopic NPTX2 expression significantly promoted G0-G1 arrest and cell apoptosis, and reduced cell proliferation, migration and invasion. Notably, the pro-apoptotic gene bax expression was significantly up-regulated while pro-survival gene bcl-2 did not significantly change in the stably transfected cells. Meanwhile, Cyclin D1 was significantly down-regulated. This study suggests that NPTX2, as a tumor-suppressor, plays an anti-tumor effect on pancreatic cancer and its low expression, due to promoter hypermethylation, may play a role in the tumorigenesis of pancreatic cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; C-Reactive Protein; Cell Line, Tumor; Cell Movement; Cell Proliferation; Collagen; Cyclin D1; DNA Methylation; Drug Combinations; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Laminin; Neoplasm Invasiveness; Nerve Tissue Proteins; Pancreas; Pancreatic Neoplasms; Proteoglycans; RNA Transport; RNA, Messenger; S Phase; Transfection

It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of β-catenin while the suppression of PAUF by shRNA down-regulates β-catenin. The induction of b-catenin by PAUF is mediated by the activities of Akt and GSK-3β, but inhibition of downstream ERK does not reduce β-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of β-catenin, we examined the phosphorylation status of β-catenin in the presence of PAUF compared with that of β-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of β-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of b-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both cyclin-D1 and c-Jun, target genes of β-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of β-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize β-catenin via a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.

Topics: Adenocarcinoma; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Lectins; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-jun; Signal Transduction; Up-Regulation

MUC1 is a transmembrane glycoprotein which is typically expressed at the apical membrane of normal epithelial cells. In cancer cells, the over-expression of MUC1 and its aberrant localization around the cell membrane and in the cytoplasm favours its interaction with different protein partners such as epidermal growth factor receptor (EGFR) and can promote tumour proliferation through the activation of oncogenic signalling pathways. Our aims were to study the mechanisms inducing MUC1 cytoplasmic localization in pancreatic cancer cells, and to decipher their impact on EGFR cellular localization and activation. Our results showed that galectin-3, an endogenous lectin, is co-expressed with MUC1 in human pancreatic ductal adenocarcinoma, and that it favours the endocytosis of MUC1 and EGFR. Depletion of galectin-3 by RNA interference increased the interaction between MUC1 and EGFR, EGFR and ERK-1,2 phosphorylation, and translocation of EGFR to the nucleus. On the contrary, silencing of galectin-3 led to a decrease of cyclin-D1 levels and of cell proliferation. The galectin-3-dependent regulation of MUC1/EGFR functions may represent an interesting mechanism modulating the EGFR-stimulated cell growth of pancreatic cancer cells.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; ErbB Receptors; Galectin 3; Humans; Mucin-1; Pancreatic Neoplasms; Protein Transport; RNA Interference

Pancreatic cancer is one of the most lethal malignancies with poor prognosis. Previously, we found that a subpopulation of cancer stem cells (CSCs) in the Panc-1 pancreatic cancer cell line could propagate to form spheres. Here we characterized the malignant phenotypes of the pancreatic cancer stem CD44+/CD24+ cells, which were enriched under sphere forming conditions as analyzed by flow cytometry. These cells demonstrated increased resistance to gemcitabine and increased migration ability. Moreover, these cells exhibited epithelial to mesenchymal transition characterized by a decreased level of the epithelial marker E-cadherin and an increased level of the mesenchymal marker vimentin. Notably, abnormal expression of Bmi-1, ABCG2, Cyclin D1 and p16 were found in Panc-1 CSCs. Our results suggest that targeted inhibition of CSCs represents a novel therapeutic approach to overcome chemoresistance and metastasis of pancreatic cancer.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cadherins; CD24 Antigen; Cell Line; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Deoxycytidine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gemcitabine; Humans; Hyaluronan Receptors; Neoplasm Proteins; Neoplastic Stem Cells; Pancreatic Neoplasms; Polycomb Repressive Complex 1; Vimentin

To evaluate the effect of RNA interference (RNAi) mediated silence of signal transduction and activation of transcription (STAT)3 on the growth of human pancreatic cancer cells both in vitro and in vivo.. STAT3 specific shRNA was used to silence the expression of STAT3 in pancreatic cancer cell line SW1990. The anti-growth effects of RNAi against STAT3 were studied in vitro and in experimental cancer xenografts in nude mice. The potential pathways involved in STAT3 signaling were detected using reverse transcription polymerase chain reaction and western blotting.. The expression of the STAT3 was inhibited using RNAi in SW1990 cells. RNAi against STAT3 inhibited cell proliferation, induced cell apoptosis and significantly reduced the levels of CyclinD1 and Bcl-xL when compared with parental and control vector-transfected cells. In vivo experiments showed that RNAi against STAT3 inhibited the tumorigenicity of SW1990 cells and significantly suppressed tumor growth when it was directly injected into tumors.. STAT3 signaling pathway plays an important role in the progression of pancreatic cancer, and silence of STAT3 gene using RNAi technique may be a novel therapeutic option for treatment of pancreatic cancer.

Topics: Animals; Apoptosis; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor

Pancreatic cancer is an aggressive malignancy with a poor prognosis. The overall 5-year survival rate of pancreatic cancer is less than 5%, and has not improved significantly for years. Further understanding of the molecular carcinogenesis of pancreatic cancer is critical for designing effective ways to treat this type of malignancy.. In this study, we examine expression of hedgehog signaling molecules in 54 surgically removed pancreatic cancer specimens as well as seven available pancreatic cancer cell lines.. We find that expression of Ptch is associated with tumor size, tumor differentiation, lymph node metastasis, and clinical stages, whereas expression of Smo is associated with tumor differentiation and lymph node metastasis. Our studies from pancreatic cancer cell lines indicate that targeted inhibition of hedgehog signaling by Smo signaling inhibitor KAAD-cyclopamine causes hedgehog target gene expression (Gli1) suppression, induces P21 expression and G1 cell population, and reduced expression of Cyclin D1 and IGF2.. These results indicate that hedgehog signaling activation is a very common event in pancreatic cancer and that targeted inhibition of hedgehog signaling may be effective in treatment of pancreatic cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cinnamates; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression; Hedgehog Proteins; Humans; Immunoenzyme Techniques; Insulin-Like Growth Factor II; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Veratrum Alkaloids

The lymphoid enhancer factor 1 (Lef-1) belongs to the nuclear transducers of canonical Wnt-signalling in embryogenesis and cancer. Lef-1 acts, in cooperation with beta-catenin, as a context-dependent transcriptional activator or repressor, thereby influencing multiple cellular functions such as proliferation, differentiation and migration. Here we report that an increased Lef-1 expression in human pancreatic cancer correlates with advanced tumour stages. In pancreatic tumours, two different transcripts of Lef-1 have been detected in various stages, as demonstrated by RT-PCR analysis. One transcript was identified as the full length Lef-1 (Lef-1 FL), whereas the second, shorter transcript lacked exon VI (Lef-1 Deltaexon VI) compared to the published sequence. Comparative analysis of these two Lef-1 variants revealed that they exhibit different cellular effects after transient expression in pancreatic carcinoma cells. Forced expression of Lef-1 Deltaexon VI inhibited E-cadherin expression in a beta-catenin-independent way. Increased amounts of Lef-1 Deltaexon VI resulted in reduced cellular aggregation and increased cell migration. Expression of Lef-1 FL, but not the newly identified Lef-1 Deltaexon VI, induced the expression of the cell cycle regulating proteins c-myc and cyclin D1 in cooperation with beta-catenin and it enhanced cell proliferation. Our findings indicate that expression of alternatively spliced Lef-1 isoforms is involved in the determination of proliferative or migratory characteristics of pancreatic carcinoma cells.

Topics: Blotting, Western; Cadherins; Carcinoma, Pancreatic Ductal; Cell Adhesion; Cell Line, Tumor; Cyclin D1; Fibronectins; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lymphoid Enhancer-Binding Factor 1; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction

The aim of this study was to assess the biological consequences of cyclin D1 silencing in pancreatic cancer cells. A replication-defective lentivirus based small hairpin RNA (shRNA) system targeting cyclin D1 caused a marked reduction in cyclin D1 protein levels in ASPC-1 and BxPC3 pancreatic cancer cell lines in conjunction with decreased cell growth and invasiveness in vitro. Moreover, a single intratumoral injection of the recombinant lentivirus targeting cyclin D1 attenuated the growth of pre-existing tumors arising from two distinct cell lines. This attenuated growth correlated with decreased proliferation and angiogenesis, as well as attenuated vascular endothelial growth factor expression. It is concluded that lentivirus-delivered shRNA targeting cyclin D1 suppresses the growth, invasiveness, tumorigenicity and pro-angiogenic potential of human pancreatic cancer cells, thereby raising the possibility that intratumoral injections of viruses targeting cyclin D1 could provide a new therapeutic approach in pancreatic ductal adenocarcinoma.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Genetic Therapy; Genetic Vectors; Humans; Immunoblotting; Immunohistochemistry; Lentivirus; Pancreatic Neoplasms

To investigate whether aspirin is able to augment gemcitabine-induced cytotoxicity in human pancreatic cancer cells.. Two gemcitabine-insensitive human pancreatic cancer cell lines, PANC-1 and Capan-1, were used. Cells were treated with either aspirin or gemcitabine alone or both of them. Cell growth and apoptosis were determined by MTT assay, Annexin V or Hoechest 33258 staining. Cell cycle distribution was examined by flow cytometry. Western blot with specific phosphorylated protein antibodies was used to detect the activation of protein kinase. RT-PCR and Western blot were applied to assess the transcription and protein level for cyclin D1 and Bcl-2.. Aspirin alone significantly inhibits the proliferation of PANC-1 cells by causing cell cycle arrest at G(1) phase. Aspirin potentiates the anti-survival effect of gemcitabine as well as its pro-apoptotic effect in PANC-1 cells, although aspirin per se does not trigger apoptosis. Aspirin inhibits GSK-3beta activation and suppresses the expression of its downstream gene products (cyclin D1 and Bcl-2), which are implicated in proliferation, survival and chemoresistance of pancreatic cancer. The effects of aspirin on Capan-1, were similar to that on PANC-1.. Our results suggest that aspirin inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells and augments the antisurvival effect of gemcitabine, probably by suppressing the activity of GSK-3beta and its downstream gene products.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Aspirin; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Deoxycytidine; Drug Synergism; Flow Cytometry; Gemcitabine; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Pancreatic Neoplasms; Proto-Oncogene Proteins c-bcl-2

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Topics: Animals; Anthraquinones; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Nude; Microtubule-Associated Proteins; Neoplasms; Pancreatic Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Sulfonamides; Survivin; Xenograft Model Antitumor Assays

The cyclin D1 (CCND1) and cyclin D3 (CCND3) are frequently co-overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here we examine their differential roles in PDAC.. CCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p < 0.005). In contrast, focal adhesion/actin cytoskeleton, MAPK and NF B signaling appeared to characterize the target genes and their interacting proteins in CCND1 suppressed PANC1 cells.. Our results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis.

Topics: Actins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin D1; Cyclin D3; Cytoskeleton; Down-Regulation; Focal Adhesions; G1 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; S Phase

Survival of patients with pancreatic cancer remains poor due to inadequate chemotherapeutic options. Sansalvamide A, a cyclic depsipeptide produced by a marine fungus, has demonstrated significant anticancer activity. We previously observed antiproliferative effects in a series of sansalvamide A analogs in pancreatic cancer cells, one of which was further evaluated in this study. Two human pancreatic cancer cell lines (AsPC-1 and CD18) were incubated with increasing concentrations (10-50 muM) of the sansalvamide analog. Cell proliferation was then measured by thymidine incorporation and cell counting, and cell cycle analysis was determined by flow cytometry. Western blot analysis was used to evaluate expression of cyclin D1, cdk4, cdk6, cyclin E, cyclin A, cdk2, and p21. Sansalvamide caused G(1) phase cell cycle arrest in both cell lines, and Western blot analyses demonstrated up-regulation of p21, down-regulation of cyclins D1, E, and A, and cdk4, consistent with G(0)/G(1) cell cycle arrest. Cumulatively the results show that Sansalvamide A attenuates pancreatic cancer cell growth and represents a potential anticancer therapy.

Topics: Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Depsipeptides; Down-Regulation; Humans; Pancreatic Neoplasms

Recent studies indicate a strong correlation of zinc transporter ZIP4 and pancreatic cancer progression; however, the underlying mechanisms are unclear. We have recently found that ZIP4 is overexpressed in pancreatic cancer. In this study, we investigated the signaling pathway through which ZIP4 regulates pancreatic cancer growth.. The expression of cyclin D1, interleukin 6 (IL-6), and signal transducer and activator of transcription 3 (STAT3) in pancreatic cancer xenografts and cells were examined by real-time PCR, Bio-Plex cytokine assay, and Western blot, respectively. The activity of cAMP response element-binding protein (CREB) is examined by a promoter activity assay.. Cyclin D1 was significantly increased in the ZIP4 overexpressing MIA PaCa-2 cells (MIA-ZIP4)-injected orthotopic xenografts and was downregulated in the ZIP4-silenced ASPC-1 (ASPC-shZIP4) group. The phosphorylation of STAT3, an upstream activator of cyclin D1, was increased in MIA-ZIP4 cells and decreased in ASPC-shZIP4 cells. IL-6, a known upstream activator for STAT3, was also found to be significantly increased in the MIA-ZIP4 cells and xenografts and decreased in the ASPC-shZIP4 group. Overexpression of ZIP4 led to a 75% increase of IL-6 promoter activity and caused increased phosphorylation of CREB.. Our study suggest that ZIP4 overexpression causes increased IL-6 transcription through CREB, which in turn activates STAT3 and leads to increased cyclin D1 expression, resulting in increased cell proliferation and tumor progression in pancreatic cancer. These results elucidated a novel pathway in ZIP4-mediated pancreatic cancer growth and suggest new therapeutic targets, including ZIP4, IL-6, and STAT3, in pancreatic cancer treatment.

Topics: Animals; Blotting, Western; Cation Transport Proteins; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Mice; Mice, Nude; Pancreatic Neoplasms; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Up-Regulation

Previous studies in our laboratory have suggested that gankyrin expression is correlated with a malignant phenotype in colorectal cancer. Here, we investigated the possible role of gankyrin in pancreatic carcinogenesis. Gankyrin expression was significantly increased in pancreatic cancer compared to non-cancerous tissues. This expression significantly enhanced cancer cell proliferation and growth in vitro and in vivo. Suppression of gankyrin downregulated cyclin A, cyclin D1, cyclin E, CDK2, CDK4, PCNA and p-Rb but upregulated p27, Rb and p53. However, gankyrin overexpression led to opposite results. Thus, gankyrin could enhance pancreatic cancer cell proliferation by promoting cell cycle progression and p53 degradation.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Phosphorylation; Proliferating Cell Nuclear Antigen; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Retinoblastoma Protein; RNA Interference; Time Factors; Transfection; Tumor Burden; Tumor Suppressor Protein p53; Up-Regulation

NF-kappaB/p65 is constitutively activated in pancreatic cancers, where it plays a critical role in the transcriptional activation of multiple cell survival genes. We have previously shown the apoptosis-inducing effects of benzyl isothiocyanate (BITC) in pancreatic cancer cells. We hypothesized that inhibition of NF-kappaB/p65 could be the mechanism of BITC-induced apoptosis. Therefore, the effect of BITC on NF-kappaB/p65 was evaluated in BxPC-3, Capan-2, and normal HPDE-6 cells by Western blotting, transcriptional and DNA-binding activity, and immunohistochemistry in the xenografted tumors. Our results reveal a remarkable decrease in the phosphorylation of NF-kappaB/p65 at Ser(536) in both BxPC-3 and Capan-2 cells by BITC treatment. The expression of NF-kappaB/p65 was downregulated significantly in BxPC-3 cells, whereas it remained unchanged in Capan-2 cells. BITC treatment caused a significant decrease in NF-kappaB transcriptional and DNA-binding activity in both BxPC-3 and Capan-2 cells. A drastic decrease was observed in the expression and reporter activity of cyclin D1 in both the cell lines. Moreover, BITC also caused a significant decrease in the expression and activity of histone deacetylase (HDAC) 1 and HDAC3 in BxPC-3 and HDAC3 in Capan-2 cells. Overexpression of HDAC1 or HDAC3 abrogated the effects of BITC. BITC treatment did not cause any change in HDAC expression in normal HPDE-6 cells. Immunohistochemical analysis of tumors from BITC-treated mice showed significantly reduced staining for NF-kappaB, cyclin D1, HDAC1, and HDAC3 compared with control. Our results suggest inhibition of HDAC1/HDAC3 by BITC as a plausible mechanism of NF-kappaB inactivation, resulting in the in vitro and in vivo growth suppression of pancreatic cancer cells.

Topics: Apoptosis; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, Reporter; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Isothiocyanates; Neoplasm Proteins; NF-kappa B; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Binding; Transcription Factor RelA; Transcription, Genetic; Xenograft Model Antitumor Assays

Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFkappaB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFkappaB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFkappaB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFkappaB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Electrophoretic Mobility Shift Assay; Electrophysiology; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Immunohistochemistry; Membrane Potential, Mitochondrial; Mice; Mice, Nude; NF-kappa B; Pancreatic Neoplasms; Polymerase Chain Reaction; Reactive Oxygen Species; RNA, Small Interfering; Sp Transcription Factors; Sp1 Transcription Factor; Sp3 Transcription Factor; Sp4 Transcription Factor; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Xenograft Model Antitumor Assays

Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-kappaB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biological Availability; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Deoxycytidine; Disease Models, Animal; Down-Regulation; Drug Synergism; Gemcitabine; Humans; Matrix Metalloproteinase 9; Mice; Nanoparticles; Neoplasm Metastasis; NF-kappa B; Pancreatic Neoplasms; Polymers; Subcutaneous Tissue; Xenograft Model Antitumor Assays

Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small-molecule MEK inhibitor, decreases MEK/ERK pathway signaling and destabilizes cyclin D1, resulting in significant anticancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores cyclin D1 expression and G(1)-S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and phosphoinositide 3-kinase mutations are unlikely to respond to the inhibition of the MEK pathway alone but will require effective inhibition of both MEK and phosphoinositide 3-kinase/AKT pathway signaling.

Topics: Alleles; Animals; Benzamides; Cell Growth Processes; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colonic Neoplasms; Cyclin D1; Diphenylamine; Extracellular Signal-Regulated MAP Kinases; Gene Knockout Techniques; HCT116 Cells; Humans; Lung Neoplasms; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mutation; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); ras Proteins

Trop2 is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas with reported low to restricted expression in normal tissues. Expression of Trop2 has been associated with increased tumor aggressiveness, metastasis and decreased patient survival, but the signaling mechanisms mediated by Trop2 are still unknown. Here, we studied the effects murine Trop2 (mTrop2) exerted on tumor cellular functions and some of the signaling mechanisms activated by this oncogene.. mTrop2 expression significantly increased tumor cell proliferation at low serum concentration, migration, foci formation and anchorage-independent growth. These in vitro characteristics translated to increased tumor growth in both subcutaneous and orthotopic pancreatic cancer murine models and also led to increased liver metastasis. mTrop2 expression also increased the levels of phosphorylated ERK1/2 mediating cell cycle progression by increasing the levels of cyclin D1 and cyclin E as well as downregulating p27. The activation of ERK was also observed in human pancreatic ductal epithelial cells and colorectal adenocarcinoma cells overexpressing human Trop2.. These findings demonstrate some of the pathogenic effects mediated by mTrop2 expression on cancer cells and the importance of targeting this cell surface glycoprotein. This study also provides the first indication of a molecular signaling pathway activated by Trop2 which has important implications for cancer cell growth and survival.

Topics: Animals; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; HCT116 Cells; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasms; Pancreatic Neoplasms; Proteins; Signal Transduction

The novel chemopreventive nitric oxide-donating aspirin (NO-ASA) prevents nearly 90% of ductal adenocarcinomas in a animal tumor model. To decipher the mechanism of this effect, we studied in BxPC-3 human pancreatic cancer cells the sequence of signaling events leading from NO-ASA treatment to cell growth inhibition. NO-ASA inhibited the growth of BxPC-3 cells (IC(50) =13 microM), by inhibiting proliferation modestly and inducing apoptosis, necrosis and G(1)/S cell cycle block. At 15 min of treatment with NO-ASA, the intracellular levels of reactive oxygen species (ROS) began increasing (peak at 8h, baseline levels by 24h). ROS activated almost immediately in a time- and concentration-dependent manner the MAPK pathways p38, ERK and JNK (their activation was abrogated by the antioxidant N-acetylcysteine). MAPK activation induced p21(cip-1), which suppressed the levels of cyclin D1 that controls the G(1)/S cell cycle transition. NO-ASA induced COX-2 expression starting 90 min after p21(cip-1) was induced. When COX-2 expression was knocked down using siRNA against cox-2, the expression of p21(cip-1) was induced by NO-ASA, regardless of the level of expression of COX-2, suggesting a marginal, if any, role for COX-2 in the growth inhibitory effect of NO-ASA. These findings along with the temporal sequence of individual changes indicate a signaling sequence that involves ROS-->MAPKs-->p21(cip-1)-->cyclin D1-->cell death. Our findings establish the critical role of ROS as proximal signaling molecules in the action of anticancer compounds and may be useful in designing mechanism-driven approaches to cancer control.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Inhibitory Concentration 50; Necrosis; Nitric Oxide; Oxidation-Reduction; Pancreatic Neoplasms; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells.. Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity.. CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor kappaB-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1kappaB2-luciferase activity.. CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor kappaB binding site in pancreatic cancer cells.

Topics: Carcinoma, Adenosquamous; Carcinoma, Islet Cell; Carcinoma, Pancreatic Ductal; CDX2 Transcription Factor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Down-Regulation; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; NF-kappa B; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Time Factors; Transcription, Genetic; Transfection

INSM1 is a zinc finger transcription factor that plays an important role in pancreatic beta-cell development. To further evaluate its role in cell fate determination, we investigated INSM1 effects on cell cycle function. The cyclin box of cyclin D1 is essential for INSM1 binding. Competitive pull-down and co-immunoprecipitation revealed that INSM1 binding to cyclin D1 interrupts its association with CDK4 and induces hypophosphorylation of the retinoblastoma protein. An inducible Tet-on system was established in Cos-7 and Panc-1 cells. Using serum starvation, we synchronized the cell cycle and subsequently induced cell cycle progression by serum stimulation. Comparison of the INSM1 induction group with the noninduced control group, INSM1 ectopic expression causes cell cycle arrest, whereas the INSM1-mediated cell cycle arrest could be reversed by cyclin D1 and CDK4 overexpression. The proline-rich N-terminal portion of INSM1 is required for cyclin D1 binding. Mutation of proline residues abolished cyclin D1 binding and also diminished its ability to induce cell cycle arrest. Cellular proliferation of Panc-1 cells was inhibited by INSM1 overexpression demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony formation, as well as tumor growth in a nude mouse model. Taken together, we provide evidence to support that INSM1 binds to cyclin D1, interrupts cell cycle signaling, and inhibits cellular proliferation.

Topics: Animals; Cell Cycle; Cell Proliferation; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cyclin D1; Cyclin-Dependent Kinase 4; DNA-Binding Proteins; Humans; Immunoprecipitation; Insulin; Pancreatic Neoplasms; Phosphorylation; Promoter Regions, Genetic; Protein Interaction Domains and Motifs; Recombinant Proteins; Repressor Proteins; Retinoblastoma Protein; RNA, Small Interfering; Saccharomyces cerevisiae; Two-Hybrid System Techniques; Zinc Fingers

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has recently shown antitumor activity in various cancer cells. Its effect on pancreatic cancer is, however, unknown and the mechanism is unclear. The study aims to investigate its antitumor activity and underlying mechanisms in human pancreatic cancer BxPC-3 and AsPC-1 cells in vitro and subcutaneous BxPC-3 xenograft tumors in mice. The MTT assay was used to evaluate cell viability, and flow cytometry and laser scanning confocal microscopy were used to detect apoptosis, for cultured cells. Pancreatic tumors were established by subcutaneous injection of BxPC-3 cells in nude BALB/c mice, and DHA was administered intraperitoneally to the mice. The size of tumors was monitored and they were harvested after the mice had been killed. Tumor sections were immunostained with an anti-Ki-67 Ab to assess the proliferation index, or stained with TUNEL to evaluate in-situ cell apoptosis. The gene expression in cells and tumors was evaluated by western blot analysis. In the cultured cells, DHA inhibited cell viability, downregulated the expression of proliferating cell nuclear antigen and cyclin D1, and upregulated p21(WAF1/CIP1); and induced apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9, in a dose-dependent manner. Similarly, in mice bearing BxPC-3 xenograft tumors, administration of DHA inhibited tumor growth in a dose-dependent manner, and modulated tumoral gene expression consistent with the in-vitro observations. This study indicates that DHA may be a potent and promising agent to combat pancreatic cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Artemisinins; bcl-2-Associated X Protein; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Humans; Ki-67 Antigen; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Xenograft Model Antitumor Assays

Von Hippel-Lindau (VHL) disease is an inherited syndrome caused by germline mutation in the VHL tumor suppressor gene predisposing to pancreatic endocrine tumors (PET). Whether these tumors derive from preexisting endocrine microadenomatosis as in multiple endocrine neoplasia type 1 (MEN1) is yet unknown. pVHL regulates hypoxia-inducible factor (HIF) that causes transcription activity of target genes like carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), and cyclin D1. Our aim was to look for overexpression of these molecules to identify precursor endocrine lesions in the pancreas of VHL patients.. Nontumoral pancreas of 18 VHL patients operated on for PET, was examined for microadenomatosis (70% of VHL patients operated on for PET. These results demonstrate that the pVHL/HIF pathway is involved very early in pancreatic endocrine tumorigenesis in this disease.

Topics: Adenoma; Adult; Antigens, CD34; Antigens, Neoplasm; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Carbonic Anhydrase IX; Carbonic Anhydrases; Chromogranin A; Cyclin D1; Female; Glucagon; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Insulin; Male; Middle Aged; Multiple Endocrine Neoplasia Type 1; Mutation; Pancreas; Pancreatectomy; Pancreatic Neoplasms; Precancerous Conditions; Vascular Endothelial Growth Factor A; von Hippel-Lindau Disease; Von Hippel-Lindau Tumor Suppressor Protein

Solid pseudopapillary tumor (SPT) of the pancreas is very rare. This study was performed to analyze the expression of Wnt signal target genes (matrix metalloproteinase-7 [MMP-7], cyclin-D1, and c-myc) and Ki-67 in resected SPTs to determine their clinicopathologic characteristics according to their expression.. From January 1995 to December 2005, 23 patients underwent pancreatic resections for SPT of the pancreas. Among 23 formalin-fixed, paraffin-embedded tissues, 12 were evaluated as a pilot study. Immunohistochemistry was performed using various detection and antigen retrieval methods to detect MMP-7, cyclin-D1, c-myc, and Ki-67. The expression of Wnt target genes was correlated with clinicopathologic features of the patients.. Solid pseudopapillary tumors of the pancreas always showed cytoplasmic/nuclear accumulation of [beta]-catenin, frequent expression of cyclin-D1, and low proliferation index. MMP-7, cyclin-D1, c-myc, and Ki-67 were not correlated with microscopic features suggesting malignant potential (P > 0.05). Tumor size was closely related to microscopic features of malignant potential and apparently has an inverse relationship with the expression of cyclin-D1 and Ki-67 (P < 0.05).. Low proliferative index and associated MMP-7 expression may cause an unpredictable clinical course in this tumor. Subtle changes in the intracellular environment, not pathologic (morphologic) changes, may elucidate the unpredictable clinical course of this tumor.

Topics: Adolescent; Adult; beta Catenin; Carcinoma, Papillary; Cyclin D1; Female; Gene Expression; Genes, myc; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Matrix Metalloproteinase 7; Middle Aged; Pancreatic Neoplasms; Pilot Projects; Wnt Proteins

To observe clinical and pathological features of pancreatic solid-pseudopapillary tumor (SPPT), and to find some useful immunohistochemical methods for its differential diagnosis.. The clinical features of 14 SPPT patients were obtained. Each case underwent microscopic observation and immunohistochemical staining. The primary antibodies were CgA, Syn, E-cadherin, beta-catenin and Cyclin D1. These results were compared with 5 pancreatic well-differentiated tumors and well-differentiated carcinomas (WET/WEC).. SPPT mainly involved young women, and the head of pancreas was the commonest location. Tumors were always in solid and cystic gross appearance. Although the tumor's borderlines seemed clear, focal infiltrations could often be identified. The histological features of SPPT were similar in some aspects to those of WET/WEC, especially the solid pattern of WET/WEC. Both of them could express CgA and Syn. But all SPPTs lost E-cadherin membranous signals, and even had some nuclear signals(5/14), while all WET/WECs remained the same staining pattern with normal pancreas cells. beta-catenin positive signals in SPPTs were located both in nuclei and plasmas. WET/WECs' positive signals were all in membranes and plasmas, but negative ones in nuclei. Perinuclear dot-like signals could also be seen in the majority cells, which were similar to normal islet cells' staining pattern. SPPTs' nuclear positive rates of Cyclin D1 were usually more than 70% (12/14). WET/WECs' rates were all lower than 30%.. Comprehensive analysis of patients' clinical, pathological features and immunohistochemistry results, including E-cadherin, beta-catenin and Cyclin D1, was helpful to the diagnosis of SPPT and its differential diagnosis of WET/WEC.

Topics: Adult; beta Catenin; Cadherins; Carcinoma, Papillary; Cyclin D1; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Young Adult

Disturbances in the expression of cell adhesion molecules in solid-pseudopapillary tumor cells were detected by immunohistochemical methods. Positive reaction to E-cadherin, catenins, and overexpression of cyclin D1 were found; these changes reflect peculiarities of morphological structure and biological behavior of the studied neoplasms.

Topics: Adolescent; Adult; Aged; Cadherins; Catenins; Cyclin D1; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Young Adult

Fibroblast growth factor receptor 1 (FGFR1) isoform IIIc enhances and FGFR1-IIIb inhibits pancreatic cancer cell growth. Nothing is presently known about the expression and regulation of human FGFR1-III isoforms. The aim of this study was to identify regulators modulating the specific expression of human FGFR1-IIIb and FGFR1-IIIc.. Parental cells, cells overexpressing FGFR1-III isoforms, and cells harboring a tetracycline-inducible cyclin D1 antisense expression vector system were used as model systems.. FGFR1-IIIb and -IIIc were coexpressed in human pancreatic cancer cells, with FGFR1-IIIc being the predominant isoform. FGFR1-IIIb mRNA expression decreased at higher cell density, whereas FGFR1-IIIc expression remained constant. Insulinlike growth factor I and epidermal growth factor induced expression of FGFR1-IIIc without altering FGFR1-IIIb. In contrast, fibroblast growth factor (FGF)1, FGF2, and FGF5 induced FGFR1-IIIc and reduced the expression of FGFR1-IIIb. Overexpression of one isoform did not alter the expression of the corresponding FGFR1-III isoform. Inhibition of cyclin D1, known to be induced by insulinlike growth factor I, epidermal growth factor, and FGF2, resulted in an inhibition of FGFR1-IIIc expression, whereas FGFR1-IIIb expression was enhanced.. This study demonstrated for the first time that FGFR1-IIIb and FGFR1-IIIc are coexpressed and that the FGFR1-III isoformsare differentially regulated by growth factors and cyclin D1.

Topics: Alternative Splicing; Base Sequence; Cell Line, Tumor; Cyclin D1; DNA Primers; Exons; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblast Growth Factor 5; Growth Substances; Humans; Insulin-Like Growth Factor I; Pancreatic Neoplasms; Protein Isoforms; Receptor, Fibroblast Growth Factor, Type 1

To analyze in solid pseudopapillary neoplasm of the pancreas (SPNP) the consequences of the deregulated Wnt pathway by studying the expression of Wnt target glutamine synthetase (GLUL), cyclin D1, and E-cadherin, which is one of the beta-catenin binding partner in cell adhesion.. The expression of cyclin D1 and GLUL was studied at the protein and/or messenger RNA levels, and the immunolocalization for E-cadherin was analyzed in 28 SPNPs screened for beta-catenin mutations. Expression of cyclin D1, GLUL, and beta-catenin was also assessed in pancreatic endocrine tumors as controls.. Cytosolic and/or nuclear accumulation of beta-catenin was observed in all tumors; an activating beta-catenin mutation was identified in 21 (91%) of 23 tumors analyzed. E-cadherin expression is lost from the membrane and is observed in intracytosolic "dotlike" structures. Whereas cyclin D1 expression is observed widely in SPNP and endocrine tumors, GLUL expression is restricted to SPNP (100%) and rare endocrine tumors (10%) displaying Wnt activation.. The activation of the Wnt/beta-catenin pathway in SPNP has 2 main consequences. First, E-cadherin expression moved from membranous to intracytoplasmic localization. Second, GLUL expression is highly correlated with Wnt/beta-catenin activation, demonstrating its faithfulness as a Wnt target gene.

Topics: Adult; beta Catenin; Cadherins; Carcinoma, Pancreatic Ductal; Cell Membrane; Cyclin D1; Cytoplasm; DNA Mutational Analysis; Female; Gene Expression; Glutamate-Ammonia Ligase; Humans; Immunohistochemistry; Male; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Wnt Proteins

Pancreatic endocrine tumors (PETs), both functioning and nonfunctioning, are usually well differentiated and progress slowly. The 2004 World Health Organization (WHO) criteria classify PETs according to clinicopathologic features and Ki-67 proliferative index. A tumor associated with poorer prognostic features may be considered "uncertain" in behavior, but the malignant classifications are reserved for tumors showing clear signs of aggressive behavior. It remains difficult to predict malignant progression in any individual PET. The cytoskeletal protein utrophin is encoded on chromosome 6q, a region frequently lost in malignant PETs. Cyclin D1 is a highly regulated mediator of the cell cycle and is frequently overexpressed in sporadic PETs. Sporadic PETs resected or biopsied from 40 patients were identified and classified using WHO criteria (19 benign/uncertain, 21 malignant). Distinctive patterns of biologic activity in unequivocally malignant PETs were demonstrated by immunohistochemistry for utrophin and cyclin D1. Utrophin localized to cell membranes (76% in malignant versus 21% in benign/uncertain PETs, P < .0006) and cyclin D1 staining showed nuclear positivity (67% in malignant versus 17% in benign/uncertain PETs, P < .003). Membranous utrophin localization was associated with significantly reduced patient survival (P = .045). Both membranous utrophin and nuclear cyclin D1 staining were also associated with higher Ki-67 proliferative indices. In our series, neither utrophin nor cyclin D1 was predictive of malignant progression in uncertain (WHO 1.2) PETs. Further studies are warranted to elucidate the role of utrophin and cyclin D1 in the malignant progression of PETs.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Disease Progression; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Utrophin

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Topics: Animals; beta Catenin; Cell Division; Cell Line, Tumor; Cyclin D1; DNA-Binding Proteins; Down-Regulation; High Mobility Group Proteins; Histone Deacetylase 1; Histone Deacetylases; Histones; Humans; Hyperinsulinism; Insulin Resistance; Insulin-Secreting Cells; Insulinoma; Kidney; Leucine Zippers; Mice; NIH 3T3 Cells; Obesity; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Structure, Tertiary; Rats; SOXD Transcription Factors; Transcription Factors; Transduction, Genetic

Periampullary cancers, the incidence of which increases gradually with industrialization, still pose a significant challenge to clinicians and researchers. Specifically, the role of cell-cycle proteins and tumor suppressor genes in these cancers is not yet clear. Recent studies have revealed that genes and proteins related to cell cycle and apoptosis regulation may be involved in pancreatic carcinogenesis.. Tissue samples were obtained from patients with periampullary cancers who underwent surgery at the National Taiwan University Hospital without receiving previous chemotherapy or radiation therapy. All periampullary cancer tissue samples were examined by a pathologist, who was unaware of the parameters to be investigated. A total of 68 patients with periampullary cancers (29 ampulla of Vater cancers (AVCs) and 39 pancreatic ductal cancers (PDCs), including various stages and histological subtypes, were enrolled. The relevant demographic and clinicopathological information was obtained from medical records.. Cell-cycle proteins, including p16, Rb, cyclin D1, p53, and E2F1, were analyzed by immunohistochemical staining. Here, significant differences were noted between AVCs and PDCs with regard to the expression of cyclin D1. This corresponded to a poor prognosis in PDCs (P < 0.05); AVCs, on the other hand, showed a relatively high survival rate. There is no obvious statistical difference between the 2 groups with regard to the expression of p16, Rb, p53, and E2F1. The study also revealed that cyclin D1 plays different roles in the carcinogenesis of AVCs and PDCs.. The expression of cyclin D1 is more often correlated with prognosis in AVCs than in PDCs, and may serve as a biomarker for the disease.

Topics: Ampulla of Vater; Biomarkers, Tumor; Chi-Square Distribution; Common Bile Duct Neoplasms; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Pancreatic Neoplasms; Predictive Value of Tests; Prognosis; Survival Rate

The pancreatic adenocarcinoma is an aggressive and devastating disease, which is characterized by invasiveness, rapid progression, and profound resistance to actual treatments, including chemotherapy and radiotherapy. At the moment, surgical resection provides the best possibility for long-term survival, but is feasible only in the minority of patients, when advanced disease chemotherapy is considered, although the effects are modest. Several studies have shown that thyroid hormone, 3,3',5-triiodo-l-thyronine (T(3)) is able to promote or inhibit cell proliferation in a cell type-dependent manner. The aim of the present study is to investigate the ability of T(3) to reduce the cell growth of the human pancreatic duct cell lines chosen, and to increase the effect of chemotherapeutic drugs at conventional concentrations. Three human cell lines hPANC-1, Capan1, and HPAC have been used as experimental models to investigate the T(3) effects on pancreatic adenocarcinoma cell proliferation. The hPANC-1 and Capan1 cell proliferation was significantly reduced, while the hormone treatment was ineffective for HPAC cells. The T(3)-dependent cell growth inhibition was also confirmed by fluorescent activated cell sorting analysis and by cell cycle-related molecule analysis. A synergic effect of T(3) and chemotherapy was demonstrated by cell kinetic experiments performed at different times and by the traditional isobologram method. We have showed that thyroid hormone T(3) and its combination with low doses of gemcitabine (dFdCyd) and cisplatin (DDP) is able to potentiate the cytotoxic action of these chemotherapic drugs. Treatment with 5-fluorouracil was, instead, largely ineffective. In conclusion, our data support the hypothesis that T(3) and its combination with dFdCyd and DDP may act in a synergic way on adenopancreatic ductal cells.

Topics: Antimetabolites; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Carcinoma, Pancreatic Ductal; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Deoxycytidine; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Fluorouracil; Gemcitabine; Humans; p21-Activated Kinases; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptors, Thyroid Hormone; Triiodothyronine

Pancreatic cancer is a highly aggressive disease that remains refractory to various chemotherapeutic agents. Because the proto-oncogene c-myc can modulate apoptosis in response to cytotoxic insults and is commonly overexpressed in pancreatic cancer, we investigated the value of c-myc as a potential modulator of cellular response to various chemotherapeutic agents.. Stable overexpression or small interfering RNA (siRNA)-mediated knockdown of c-myc and restoration of cyclin D1 were done in the Ela-myc pancreatic tumor cell line. Cell viability after cisplatin treatment of c-myc-overexpressing, control, and siRNA-transfected cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and drug-induced apoptosis was measured by DNA fragmentation, sub-G(1), and poly(ADP-ribose) polymerase cleavage analyses. Protein expression profile after cisplatin treatment was determined by Western blotting and DNA binding activity of nuclear factor-kappaB was examined by electrophoretic mobility shift assay.. Ectopic overexpression of c-myc in murine and human pancreatic cancer cell lines, Ela-myc and L3.6pl, respectively, resulted in increased sensitivity to cisplatin and other chemotherapeutic drugs. Increased sensitivity to cisplatin in c-myc-overexpressing cells was due, in part, to the marked increase in cisplatin-induced apoptosis. Conversely, down-regulation of c-myc expression in stable c-myc-overexpressing cells by c-myc siRNA resulted in decreased sensitivity to cisplatin-induced cell death. These results indicate an important role of c-myc in chemosensitivity of pancreatic cancer cells. The c-myc-induced cisplatin sensitivity correlated with inhibition of nuclear factor kappaB activity, which was partially restored by ectopic cyclin D1 overexpression.. Our results suggest that the c-myc-dependent sensitization to chemotherapy-induced apoptosis involves suppression of cyclin D1 expression and nuclear factor kappaB activity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Cisplatin; Cyclin D1; DNA Fragmentation; Drug Resistance, Neoplasm; Humans; Mice; NF-kappa B; Pancreatic Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; RNA, Small Interfering

The HMGA1 proteins act as architectural transcription factors and are involved in the regulation of genes important in the process of carcinogenesis. Although HMGA1 proteins are overexpressed in most types of cancer, signaling circuits regulated by HMGA1 are not clarified in detail. In this study, we show that HMGA1 proteins promote proliferation of pancreatic cancer cells by accelerating G(1) phase progression. Transfection of HMGA1-specific small interfering RNA (siRNA) activates the RB-dependent G(1)-phase checkpoint due to the impaired expression of cyclin D1. Down-regulation of cyclin D1 after the HMGA1 knockdown is due to translational control and involves the repressor of the eukaryotic translation initiation factor 4E (eIF4E) 4E-BP1. We show that 4E-BP1 and cyclin D1 act downstream of the insulin receptor (IR) in pancreatic cancer cells. At the molecular level transcription of the IR is controlled by a CAAT/enhancer binding protein beta (C/EBPbeta)/HMGA1 complex. Together, this work defines a novel pathway regulated by HMGA1, which contributes to the proliferation of pancreatic cancer cells.

Topics: Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; HMGA1a Protein; Humans; Pancreatic Neoplasms; Protein Biosynthesis; Receptor, Insulin; RNA Interference; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The design of novel targeted or combination therapies may improve treatment options for pancreatic cancer. Two targets of recent interest are nuclear factor-kappaB (NF-kappaB) and cyclooxygenase (COX), known to be activated or overexpressed, respectively, in pancreatic cancer. We have previously shown that parthenolide, a proapoptotic drug associated with NF-kappaB inhibition, enhanced the growth suppression of pancreatic cancer cells by the COX inhibitor sulindac in vitro. In the present study, a bioavailable analogue of parthenolide, LC-1, and sulindac were evaluated in vivo using a xenograft model of human pancreatic cancer. Treatment groups included placebo, low-dose/high-dose LC-1 (20 and 40 mg/kg), low-dose/high-dose sulindac (20 and 60 mg/kg), and low-dose combination LC-1/sulindac (20 mg/kg each). In MiaPaCa-2 xenografts, tumor growth was inhibited by either high-dose sulindac or LC-1. In BxPC-3 xenografts, tumor size was significantly reduced by treatment with the low-dose LC-1/sulindac combination or high-dose sulindac alone (P < 0.05). Immunohistochemistry of BxPC-3 tumors revealed a significant decrease in Ki-67 and CD31 staining by high-dose sulindac, with no significant changes in COX-1/COX-2 levels or activity in any of the treatment groups. NF-kappaB DNA-binding activity was significantly decreased by high-dose LC-1. Cyclin D1 protein levels were reduced by the low-dose LC-1/sulindac combination or high-dose sulindac alone, correlating with BxPC-3 tumor suppression. These results suggest that LC-1 and sulindac may mediate their antitumor effects, in part, by altering cyclin D1 levels. Furthermore, this study provides preclinical evidence for the therapeutic efficacy of these agents.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cyclin D1; Humans; Mice; Mice, Nude; NF-kappa B; Pancreatic Neoplasms; Prostaglandin-Endoperoxide Synthases; Sesquiterpenes; Sulindac

Abrogation of the Wnt-signaling pathway is implicated in the carcinogenesis of several malignancies, especially colorectal cancer where up to 90% of cases are thought to have impaired Wnt signaling. It is less frequently involved in conventional ductal pancreatic adenocarcinoma. This pathway has not been explored in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas previously and formed the basis of this study. A tissue microarray of 18 cases of IPMN was stained for proteins involved in the Wnt pathway: adenomatous polyposis coli (APC), pan-beta-catenin, axin 2, glycogen synthase 3alphabeta and 3beta, c-myc, E-cadherin, and cyclin D1. The IPMNs were classified as 8 adenomas, 3 borderline, and 7 cases with carcinoma in situ and/or invasive carcinoma, occurring in 13 females, and the overall age range was 45 to 73 years. Immunohistochemical analysis showed nuclear beta-catenin staining in 7 (39%) of the 18 cases. The cases with nuclear beta-catenin localization included 1 adenoma, 2 borderline IPMN, and 4 carcinomas in situ and/or invasive carcinomas. Seven cases showed absence of APC immunostaining and these included 4 cases with nuclear beta-catenin localization. Fourteen cases displayed marked diffuse up-regulation of c-myc protein, and 12 cases also showed diffuse cyclin D1 protein overexpression. E-cadherin expression was intense and membrane in location (comparable to normal tissue) in 6 of 8 adenomas (no tissue was available in 1 case). Decreased E-cadherin staining was noted in 8 cases where tissue was available for assessment. There was progressive decrease in membrane staining of E-cadherin in 2 of 3 borderline lesions, 1 of 2 carcinomas in situ, and 4 of 5 invasive carcinomas. All other immunostains were either normal in distribution or did not show any correlation with beta-catenin or clinicopathologic parameters. In conclusion, 7 (39%) of 18 cases of IPMN in this study demonstrated abnormal localization of beta-catenin, 4 of which also lacked APC expression. Of 5 carcinomas arising in IPMN, 4 displayed a decrease in E-cadherin expression. There was also a trend for the higher grades of IPMN to show nuclear localization of beta-catenin. These findings suggest that a proportion of cases of IPMN may show abnormalities in the Wnt-signaling pathway with consequent altered expression of downstream related proteins.

Topics: Adenoma; Aged; beta Catenin; Cadherins; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cyclin D1; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Protein Array Analysis; Proto-Oncogene Proteins c-myc; Wnt Proteins

The epidermal growth factor receptor (EGFR) signaling network plays critical roles in human cancers, including pancreatic cancer, suggesting that the discovery of specific agents targeting EGFR would be extremely valuable for pancreatic cancer therapy. EGFR-related protein (ERRP), a recently identified pan-erbB inhibitor, has been shown to inhibit growth and induce apoptosis of pancreatic cancer cells in vitro and tumor growth in a xenograft model. However, the precise molecular mechanism(s) by which ERRP exerts its antitumor activity remains unclear. The current investigation was undertaken to delineate the tumor growth inhibitory mechanism(s) of ERRP in pancreatic cancer cells. Using multiple molecular assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, apoptosis, gene transfection, real-time reverse transcription-PCR, Western blotting, invasion, and electrophoretic mobility shift assay for measuring DNA-binding activity of nuclear factor-kappaB (NF-kappaB), we found that ERRP caused marked inhibition of pancreatic cancer cell growth. This was accompanied by increased apoptosis and concomitant attenuation of Notch-1 and NF-kappaB and down-regulation of NF-kappaB downstream genes, such as matrix metalloproteinase-9 and vascular endothelial growth factor, resulting in the inhibition of pancreatic cancer cell invasion through the Matrigel. We also found that down-regulation of Notch-1 by small interfering RNA before ERRP treatment resulted in enhanced cell growth inhibition and apoptosis. Our data suggest that the ERRP-mediated inactivation of EGFR, Notch-1, NF-kappaB, and its downstream target genes contributed to the inhibition of cell growth and invasion. We conclude that ERRP could be an effective agent for inhibiting tumor growth and invasion for the treatment of pancreatic cancer.

Topics: Apoptosis; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; ErbB Receptors; Glycoproteins; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; NF-kappa B; Pancreatic Neoplasms; Receptor, Notch1; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Vascular Endothelial Growth Factor A

Solid pseudopapillary tumor (SPT) is an unusual pancreatic neoplasm of low malignant potential that most frequently occurs in young women. The tumor is indolent, with long patient survival, even in the presence of extension into adjacent organs and metastases. Histologically, it is a solid and cystic tumor with a prominent vascular network and degenerative pseudopapillae formation. Despite its distinctive morphology and cytological features, its histogenesis is unclear. Herein, we report a case of solid pseudopapillary tumor in a 41-year-old female in which the tumor cells immunohistochemically and ultrastructurally suggest a centroacinar cell origin. The tumor cells and the normal centroacinar cells stained positive for alpha-antitrypsin (alpha-AT), CD10, cyclin D1 and NSE. Ultrastructural examination shows similarities in nuclear shape, nucleoli location and cytoplasmic contents between neoplastic cells and normal centroacinar cells of the pancreas. Based on both immunohistochemical and ultrastructural features, we propose that the centroacinar cell is the origin of SPT.

Topics: Adult; alpha 1-Antitrypsin; Carcinoma, Papillary; Cyclin D1; Female; Humans; Islets of Langerhans; Neprilysin; Pancreatic Neoplasms; Phosphopyruvate Hydratase

Herein, we show that the hematopoietic-specific GEF VAV1 is ectopically expressed in primary pancreatic adenocarcinomas due to demethylation of the gene promoter. Interestingly, VAV1-positive tumors had a worse survival rate compared to VAV1-negative tumors. Surprisingly, even in the presence of oncogenic KRAS, VAV1 RNAi abrogates neoplastic cellular proliferation in vitro and in vivo, thus identifying Vav1 as a growth-stimulatory protein in this disease. Vav1 acts synergistically with the EGF receptor to stimulate pancreatic tumor cell proliferation. Mechanistically, the effects of Vav1 require its GEF activity and the activation of Rac1, PAK1, and NF-kappaB and involve cyclin D1 upregulation. Thus, the discovery of prooncogenic pathways regulated by Vav1 makes it an attractive target for therapeutic intervention.

Topics: Adenocarcinoma; Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; DNA Methylation; Epidermal Growth Factor; Humans; Male; Mice; Mice, Nude; p21-Activated Kinases; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Signal Transduction; Survival Rate

BCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies.. Regulation of BCL-2 gene transcription was examined in seven different human cancer cell lines including two pancreatic (MIA-PaCa-2, PANC-1), two prostate (LNCaP, PC-3), two lung (Calu-1, A549) and one breast (MCF-7) cancer cell line. Cells were treated with inhibitors of phosphatidylinositol-3 kinase (PI3K), MEK/ERK, and p38MAPK. The effect of mutation of a NF-kappaB site in the BCL-2 promoter was determined, as was the effect of inhibition of NF-kappaB function using a 26S proteasome inhibitor (bortezomib) on both BCL-2 transcription and induction of apoptosis.. BCL-2 expression varied both between and within tumor types; four of seven cell lines demonstrated high BCL-2 levels (MIA-PaCa-2, PC-3, Calu-1 and MCF-7). No signaling pathway was uniformly responsible for overexpression of BCL-2; however, mutation of the NF-kappaB site decreased BCL-2 promoter activity in all cell lines. Inhibition of NF-kappaB activity decreased BCL-2 protein levels independently of the signaling pathway involved in transcriptional activation of the BCL-2 gene.. Diverse signaling pathways variably regulate BCL-2 gene expression in a cell type-specific fashion. Therapy to decrease BCL-2 levels in various human cancers would be more broadly applicable if targeted to transcriptional activation rather than signal transduction cascades. Finally, the apoptotic efficacy of proteasome inhibition with bortezomib paralleled the ability to inhibit NF-kappaB activity and decrease BCL-2 levels.

Topics: Boronic Acids; Bortezomib; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Male; NF-kappa B; Pancreatic Neoplasms; Prostatic Neoplasms; Protease Inhibitors; Pyrazines; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

The Ras-association domain family 1A (RASSF1A) tumour suppressor gene is inactivated in a variety of solid tumours, usually by epigenetic silencing of the promoter and/or allelic loss of its locus at 3p21.3. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, 62 endocrine tumours from different sites in the gut were investigated for methylation of the RASSF1A promoter using the polymerase chain reaction, the presence of 3p21.3 deletions by loss of heterozygosity analysis, and cyclin D1 expression by immunohistochemistry. Methylation was found in 20/62 (32%) cases and was restricted to foregut tumours; deletion at 3p21.3 was found in 15/58 (26%) informative cases and restricted to malignant foregut tumours; cyclin D1 hyper-expression was found in 31/58 (53%) cases and correlated with RASSF1A methylation. Our data suggest that RASSF1A is involved in the development of endocrine tumours derived from the foregut only, and that the presence of both RASSF1A methylation and 3p21.3 deletion is associated with malignancy. These results may provide a rationale for foregut-targeted therapy for aggressive endocrine carcinomas entailing the use of demethylating agents.

Topics: Adult; Aged; Aged, 80 and over; Appendiceal Neoplasms; Carcinoma, Neuroendocrine; Cyclin D1; Duodenal Neoplasms; Female; Gastrointestinal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Ileal Neoplasms; Intestinal Neoplasms; Loss of Heterozygosity; Male; Methylation; Middle Aged; Pancreatic Neoplasms; Promoter Regions, Genetic; Rectal Neoplasms; Stomach Neoplasms; Tumor Suppressor Proteins

To investigate the effect of baicalin on the proliferation of insulinoma cell line and the molecular mechanism involved.. Such methods as light microscope, MTT assay, flow cytometry and Western blotting were applied to investigate the effects of baicalin (0, 100, 200, and 400 microg/ml baicalin treated for 24 h or 200 microg/ml baicalin treated at different time points) on the cell proliferation, cell survival rate, the cell cycle and related molecular mechanisms.. The number of proliferating cells obviously decreased with the increase of baicalin under the light microscope, and the survival rate of cells decreased as determined by MTT assay. After being treated with baicalin, the number of insulinoma cells in S-phase obviously decreased from 38.2% (0 microg/ml) to 9.4% (400 microg/ml), and the number of cells in phase G1 increased from 56.4% (0 microg/ml) to 85.9% (400 microg/ml). In the meantime, the expression of cyclin D1 was obviously declined by Western blotting.. Baica-lin can inhibit the proliferation of insulinoma cells, and the down-regulation of the expression of cyclin D1 might also be involved in these events.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flavonoids; Humans; Insulinoma; Pancreatic Neoplasms

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Mice; Mice, Transgenic; Pancreatic Neoplasms

Elevated cyclin D1 in human pancreatic cancer correlates with poor prognosis. Because pancreatic cancer is invariably resistant to chemotherapy, the goal of this study was to examine whether the drug resistance of pancreatic cancer cells is in part attributed to cyclin D1 overexpression.. Stable overexpression and small interfering RNA (siRNA)--mediated knockdown of cyclin D1 were done in the newly established Ela-myc pancreatic tumor cell line. Cisplatin sensitivity of control, overexpressing, and siRNA-transfected cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clonogenic, and apoptotic assays [DNA fragmentation, sub-G1, and poly(ADP-ribose) polymerase cleavage analysis]. The role of nuclear factor-kappaB and apoptotic proteins in cyclin D1-mediated chemoresistance was examined by EMSA and Western blotting, respectively.. Overexpression of cyclin D1 in Ela-myc pancreatic tumor cells promoted cell proliferation and anchorage-independent growth. Moreover, cyclin D1-overexpressing cells exhibited significantly reduced chemosensitivity and a higher survival rate upon cisplatin treatment, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, respectively. Although overexpression of cyclin D1 rendered cells more resistant to cisplatin-induced apoptosis, siRNA-directed suppression of cyclin D1 expression resulted in enhanced susceptibility to cisplatin-mediated apoptosis. The attenuation of cisplatin-induced cell death in cyclin D1-overexpressing cells was correlated with the up-regulation of nuclear factor-kappaB activity and maintenance of bcl-2 and bcl-xl protein levels.. These results suggest that overexpression of cyclin D1 can contribute to chemoresistance of pancreatic cancer cells because of the dual roles of cyclin D1 in promoting cell proliferation and in inhibiting drug-induced apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Culture Media; Cyclin D1; Deoxycytidine; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Electrophoretic Mobility Shift Assay; Female; Flow Cytometry; Gemcitabine; Gene Expression Regulation, Neoplastic; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; NF-kappa B; Pancreatic Elastase; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection

Retinoids may be useful agents for the treatment of pancreatic cancer. However, retinoic acid receptor (RAR)-selective retinoids produce unwanted side effects. In contrast, retinoid X receptor (RXR)-selective retinoids produce fewer side effects; however, it was not known whether RXR-selective retinoids could reduce pancreatic tumor cell proliferation. In the present study, the novel RXR-selective retinoid, AGN194204, was compared with that of other retinoids for the ability to suppress pancreatic cancer cell proliferation. We treated various pancreatic cancer cell lines with receptor-selective ligands and cytotoxic agents and monitored the effects on cell proliferation, markers of apoptosis and cell cycle. Our results indicate that AGN194204, at concentrations >10 nM, inhibits proliferation of MIA PaCa-2 and BxPC-3 cells but not the proliferation of AsPC-1 cells. Moreover, in BxPC-3 and MIA PaCa-2 cells, AGN194204 was 10-100 times more effective than RAR-selective retinoids. AGN194204-dependent suppression of MIA PaCa-2 cell proliferation is associated with reduced cyclin E and cyclin-dependent kinase 6 (cdk6) level, but cyclin D1, cdk2 and cdk4 content is not altered. In addition, p27 level increases 2-fold. The RXR-selective antagonist, AGN195393, reverses the AGN194204-dependent growth inhibition and the decline in cyclin E and cdk6 levels. In contrast, these changes are not reversed by treatment with the RAR antagonist, AGN193109. AGN194204 did not appear to alter cell apoptosis as measured by change in cleavage of procaspase-3, -8 or -9. We also examined the effects AGN194204 co-treatment with cytotoxic agents. Treatment of MIA PaCa-2 cells with AGN194204 + cisplatin, gemcitabine, 5-fluorouracil, interferon (IFN)alpha or IFNgamma resulted in an additive but not synergistic reduction in MIA PaCa-2 cell number. These results indicate that AGN194204, an RXR-selective retinoid, is a more effective inhibitor of pancreatic cell proliferation than the RAR-selective retinoids, and further indicate that AGN194204 produces an additive reduction in cell number when given with other agents. Our results suggest that RXR-selective ligands, which are less toxic than RAR-selective ligands, may be suitable agents for the treatment of pancreatic cancer.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; Deoxycytidine; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Fluorouracil; Gemcitabine; Humans; Interferon-alpha; Interferon-gamma; Ligands; Pancreatic Neoplasms; Plasmids; Protein Binding; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Tetrahydronaphthalenes; Time Factors; Transcription Factors

Benzyl isothiocyanate (BITC), a cruciferous vegetable-derived compound, has been shown to inhibit chemically induced cancer in animal models. Moreover, epidemiological studies have provided compelling evidence to suggest that cruciferous vegetables may be protective against cancer risk. Here, we report that BITC significantly inhibits growth of human pancreatic cancer BxPC-3 cells in a concentration-dependent manner with an IC(50) of approximately 8 micro M, a concentration that can be generated through dietary intake of cruciferous vegetables. Treatment of BxPC-3 cells with growth suppressive concentrations of BITC resulted in G(2)/M phase cell cycle arrest that was associated with a marked decline in protein levels of G(2)/M regulatory proteins including cyclin-dependent kinase 1 (Cdk1), cyclin B1 and cell division cycle 25B (Cdc25B). Further, BITC-mediated growth inhibition of BxPC-3 cells correlated with apoptosis induction that was characterized by an increase in Bax/Bcl-2 ratio, cleavage of procaspase-3 and poly(ADP-ribose)polymerase (PARP), and an increase in cytoplasmic histone-associated DNA fragmentation. Interestingly, BITC treatment caused inhibition of nuclear factor kappaB (NF-kappaB) activation, which is constitutively activated in human pancreatic cancer. Western blotting revealed concentration-dependent decrease in NF-kappaB/Rel-p65 protein level in BxPC-3 cells upon exposure to BITC. An increase in protein level of inhibitory subunit kappaB (IkappaBa) in association with reduced serine-32 phosphorylation was also observed in BITC-treated BxPC-3 cells. Consistent with these findings, BITC treatment caused a decrease in nuclear translocation of NF-kappaB as reflected by reduced DNA-binding capacity of NF-kappaB. Furthermore, the protein level of cyclin D1, a transcriptional target of NF-kappaB, was reduced significantly in BITC-treated BxPC-3 cells. To the best of our knowledge, this study is the first published report to implicate suppression of NF-kappaB activation as a potential mechanism for anti-proliferative activity of BITC against human pancreatic cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cyclin B; Cyclin B1; Cyclin D1; Humans; Isothiocyanates; NF-kappa B; Pancreatic Neoplasms; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

To observe the growth effect of somatostapin on human pancreas cancer lines Bxpc-3.. The Bxpc-3 pancreas cancer cells were treated with Somatotropin. The cells hyperplasia were detected by MTT and were observed apoptosis cells determinated quantitatively by TUNEL, quantify immune fluoresence double marked the proliferation cells and apoptosis cells, the expression of Cyclin D1 detected by immunohistochemical.. The growth effect of pancrea cancer cells were limited by 10(-7) M, 10(-8) M, 10(-9) M Somatotropin on 2 day. The limited effect was decreased from 3 day. The cells proliferation were increased by somotostapin on 4day to 5day. The relationship between the expression of Cyclin D1 and apoptosis was negative correlation and the cells hyperplasia was positive correlation in Bxpc-3 cell line.. From the cell study we knew the expression of Cyclin D1 reflected the prolefiration of pancreas cancer cells.

Topics: Apoptosis; Cell Division; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Growth Hormone; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Pancreatic Neoplasms; Time Factors

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.

Topics: Aged; Aged, 80 and over; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 20; Consensus Sequence; Cyclin D1; ErbB Receptors; Female; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Liver Neoplasms; Male; MAP Kinase Kinase Kinases; Middle Aged; Mitogen-Activated Protein Kinase Kinase Kinase 11; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Trans-Activators

Solid pseudopapillary tumor of the pancreas (SPT) is an uncommon neoplasm of low malignant potential, generally occurring in young women. The tumor is indolent, usually with long survival, even in the presence of extension into adjacent organs and metastases. Pathological features include solid, cellular, and cystic regions and degenerative pseudopapillae formation. Despite its distinctive morphology and cytological features, the cell lineage of this entity is unclear. Here we report a case of solid pseudopapillary tumor in a 48-year-old man with 10-year follow-up in which melanin pigment was found within the tumor cells. The tumor cells stained positive not only for melanocytic markers including S-100, HMB-45, and Fontana, but also other well-established markers for this kind of neoplasm such as alpha-antitrypsin (Alpha-AT), anti-alpha-chymotrypsin (AACT), NSE, CD10, cyclin D1, and beta-catenin. Electron microscopy confirmed the formation of premelanosomes and melanosome granules in the tumor cells. To our knowledge, this is the first report in which melanosomes were produced by SPT. Because melanocytes are derived from neurocrest, we hypothesize that the histogenesis of SPT is of neurocrest origin. This phenomenon may also be explained by ongoing research in which it has been shown that Wnt signaling/beta-catenin intranuclear localization promotes pigment cell formation by medial crest cells in embryos.

Topics: Antibodies; beta Catenin; Cell Differentiation; Cyclin D1; Cytoskeletal Proteins; Humans; Immunohistochemistry; Male; Melanins; Melanocytes; Melanosomes; Middle Aged; Neprilysin; Pancreatic Neoplasms; Trans-Activators

The aim of this study was to evaluate the role of cyclin D1 and Ki67 proteins involved in cell-cycle control as a prognostic factor in pancreatic carcinomas. We examined formalin-fixed, paraffin-embedded material from 59 pancreatic adenocarcinomas, for which appropriate clinical and prognostic data were available. The standard streptavidin biotin immunoperoxidase method was used for immunostaining with cyclin D1 and Ki67. The extent of positive nuclear and cytoplasmic cyclin D1 staining was graded semiquantitatively. Ki67 reactivity was quantified and expressed as the percentage of stained nuclei. Staining with cyclin D1 and Ki67 was compared with histopathological prognostic features, and their relation with survival was also tested statistically. Patients whose tumors were cyclin D1-positive showed perineural invasion significantly more frequently than did patients with cyclin D1-negative tumors at the immunohistochemical level. In addition, tumors with lymphatic vessel invasion and without showed a significant difference in terms of cytoplasmic cyclin D1 staining. Ki67 indices were statistically different in stage groups. There was a significant and direct correlation between Ki67 index and nuclear cyclin D1 staining scores. No relation with survival was found. Our results suggest that cell-cycle proteins do not directly affect the prognosis of patients with pancreatic adenocarcinoma. Conversely, cyclin D1-positive tumors tend to have perineural invasion more frequently. In addition, lymph vessel invasion is another factor related to cyclin D1 reactivity of the cells. Ki67 indices differ statistically in stage groups.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Female; History, 16th Century; History, 17th Century; Humans; Immunohistochemistry; Ki-67 Antigen; Lymphatic Metastasis; Male; Pancreatic Neoplasms; Prognosis; Survival Analysis

Molecular analysis of pancreatic intraepithelial neoplasia lesions and ductal adenocarcinoma suggested a multistage paradigm for pancreatic duct cell carcinogenesis. This study investigated the molecular basis for the neoplastic duct cells in this pancreatic intraepithelial neoplasia-carcinoma sequence to acquire progressive enhancement of their proliferative potential.. Using tissue microarray blocks containing 15 to 40 pancreatic intraepithelial neoplasia lesions and ductal adenocarcinoma of pancreas, we studied by immunohistochemistry the expression profiles of cyclins and cyclin dependent kinases (CDKs) that regulate the G1-S cell cycle checkpoints. The role of cyclins D3 and D1 in three pancreatic cancer cell lines was investigated using specific short interfering RNA technique.. Cyclin D3 overexpression was noted the earliest in pancreatic intraepithelial neoplasia-1A and was prevalent in 90% to 100% of high-grade pancreatic intraepithelial neoplasias and ductal cancer. Cyclin A overexpression was also noted early and reached 50% to 100% of high-grade pancreatic intraepithelial neoplasias and cancer, but the percentage of abnormal duct cells showing overexpression of cyclin A was significantly lower than cyclin D3. Cyclin E overexpression occurred in 20% to 25% of high-grade pancreatic intraepithelial neoplasias and in 75% of ductal carcinoma. Cyclin D1 demonstrated the lowest frequency of overexpression that occurred late. CDK2 and CDK4 overexpression was also noted in early pancreatic intraepithelial neoplasias and progressively increased to reach 60% to 75% in carcinoma. The down-regulation of cyclin D3 mRNA and protein levels using specific short interfering RNA resulted in growth inhibition of pancreatic cancer cell lines.. The results provide additional insight into the mechanism of G1-S cell cycle checkpoints deregulation during stepwise pancreatic duct cell carcinogenesis, and suggest a p16-independent role for cyclin D3 in deregulating the G1 cell cycle checkpoints during early stages of pancreatic duct cell carcinogenesis.

Topics: Blotting, Western; CDC2-CDC28 Kinases; Cell Division; Cell Line, Tumor; Cyclin A; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Pancreatic Ducts; Pancreatic Neoplasms; Proto-Oncogene Proteins; RNA, Messenger; RNA, Small Interfering; S Phase; Tissue Array Analysis; Transfection

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.

Topics: Animals; Benzamides; Blotting, Western; Butadienes; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Extracts; Cell Line, Tumor; Cells, Cultured; Chromones; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Humans; Mice; Microscopy, Fluorescence; Morpholines; NIH 3T3 Cells; Nitriles; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; raf Kinases; Retroviridae; RNA, Messenger; Tumor Suppressor Proteins

This study concerns the significance of aberrant (nuclear/cytoplasmic) expression of beta-catenin in pancreatoblastoma (PBL). On immunohistochemistry, all seven PBLs examined showed nuclear/cytoplasmic expression of beta-catenin, predominantly in the squamoid corpuscles (SCs). In areas with acinar/ductular differentiation, few tumour cells displayed nuclear/cytoplasmic expression of beta-catenin and more than half of the tumour cells showed membranous expression. Two out of five (40%) tumours examined showed missense mutations in codons 33 and 37 of exon 3 of the beta-catenin gene. No mutation of the adenomatous polyposis coli (APC) gene was detected in two of the remaining three tumours. Amplifiable DNA for APC analysis was not obtained from the one other tumour. Immunoreactivity for cyclin D1, one of the nuclear targets of beta-catenin, was found predominantly in the SCs of the seven tumours. In contrast, the Ki-67 labelling index was 2-4% (median 3%) in the SCs and 8-18% (median 12%) in the other areas, indicating a negative correlation with nuclear cyclin D1 reactivity. These results imply that in PBLs, nuclear/cytoplasmic accumulation of beta-catenin and overexpression of its target gene cyclin D1 are not associated with the induction of tumour cell proliferation. Nuclear/cytoplasmic accumulation of beta-catenin may be related to the morphogenesis of the SCs that are considered most characteristic for PBL.

Topics: beta Catenin; Child; Child, Preschool; Cyclin D1; Cytoskeletal Proteins; DNA, Neoplasm; Female; Genes, APC; Humans; Immunohistochemistry; Male; Mutation; Neoplasm Proteins; Pancreatic Neoplasms; Sequence Analysis, DNA; Trans-Activators

Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand activated transcription factor, forms a heterodimer with retinoid X receptor alpha (RXRalpha), and its transcriptional activity is thought to be maximal in the presence of both PPARgamma and RXRalpha ligands. Though previous studies suggested that thiazolidinediones (TZDs), known as PPARgamma ligands, inhibit the growth of certain types of cancer cells, little is known about the growth inhibitory effects mediated though activation of PPARgamma/RXRalpha. We examined the effects of troglitazone (one type of TZDs) and 9-cis retinoic acid, a RXRalpha ligand, on activation of PPARgamma/RXRalpha and growth inhibition of human pancreatic cancer cell lines (AsPC1, BxPC3, PSN1, PCI6, Panc1, KMP-4, and KMP-7). Combined treatment of troglitazone and 9-cis retinoic acid showed enhanced transcriptional activity and enhanced antiproliferative effects. In PSN1 cells, G1 cell cycle arrest and apoptosis were induced by troglitazone and these effects were enhanced with additional 9-cis RA. Our findings suggest that activation of PPARgamma/RXRalpha pathway might play an important role in growth inhibition of pancreatic cancer cells via G1 cell cycle arrest and apoptosis. This nuclear receptor might be a suitable molecular target for treatment of pancreatic cancers.

Topics: Alitretinoin; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Cell Division; Chromans; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Nuclear Proteins; Pancreatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Tretinoin; Troglitazone; Tumor Cells, Cultured

To study the relationship between the abnormal expression of beta-catenin (beta-cat) and the high expressions of cyclin D1 and c-myc and the occurance, proliferation, infiltration, metastasis and prognosis of pancreatic cancer, and to provide rational basis for the clinical diagnosis and treatment.. Immunohistochemical PicTure trade mark was used to examine the expressions of beta-cat, cyclin D1 and c-myc in 47 cases of the cancerous tissue of pancreas, 12 cases of the pancreatic intraepithelial neoplasia and 10 cases of normal tissue of pancreas, respectively. Pancreatic cancer proliferation cell nuclear antigen (PCNA) was also tested as the index of the extent of proliferation of the pancreatic cancer.. beta-cat was expressed normally in the 10 cases of the normal pancreatic tissue, while cyclin D1 and c-myc were negative. The expression rates of beta-cat, cyclin D1 and c-myc in the tissues of the pancreatic intraepithelial neoplasia and the pancreatic cancer had no significant difference [6/12 and 68.1% (32/47), 6/12 and 74.5% (35/47), 5/12 and 70.2% (33/47) respectively;P values were all more than 0.05]. The abnormal expression rate of beta-cat was significantly correlated to the metastasis of the pancreatic cancer and the one-year survival rate (both P < 0.05), but had no relation with the size, the extent of differentiation, the activity of proliferation, or infiltration of the pancreatic cancer (both P > 0.05). The expression rate of cyclin D1 was correlated with the proliferation of the pancreatic cancer and the extent of differentiation (both P < 0.05), but not with the size, infiltration, metastasis, or one-year survival rate of the pancreatic cancer (both P > 0.05). The expression rate of c-myc was not correlated with the size, the extent of proliferation, infiltration, metastasis, or one-year survival rate (both P > 0.05), but closely with the proliferation activity of the cancerous tissue of pancreas (P < 0.05). The abnormal expression of beta-cat and the high expressions of cyclin D1 and c-myc had a parallel relationship with the pancreatic intraepithelial neoplasia and pancreatic cancer (both P < 0.05, gamma = 1.000, 0.845, 0.437, 0.452).. The abnormal expression of beta-cat activates cyclin D1 and c-myc, and results in the unchecked proliferation and differentiation, which may play an important role in the genesis of the pancreatic cancer. The abnormal expression of beta-cat is one of the mechanisms for the spread of pancreatic cancer and an index in the molecular biology to determine the metastasis and prognosis of pancreatic cancer.

Topics: Adult; Aged; beta Catenin; Cyclin D1; Cytoskeletal Proteins; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreas; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Trans-Activators

Pancreatic endocrine tumours (PETs) occur sporadically or are inherited as part of the multiple endocrine neoplasia type-1 syndrome. Little is known about the molecular events leading to these tumours. Cyclin D1, a key regulator of the G1/S transition of the cell cycle, is overexpressed in a variety of human cancers as well as certain endocrine tumours. We hypothesized that similar to other endocrine tumours, cyclin D1 is overexpressed in human sporadic PETs. Cyclin D1 protein overexpression was found in 20 of 31 PETs (65%) when compared with normal pancreatic tIssue. Furthermore, Northern blot analysis suggests that cyclin D1 up-regulation occurs at the post-transcriptional level in some PETs. Because the key cell growth signalling pathways p42/p44/ERK (extracellular signal-regulated kinase), p38/MAPK (mitogen-activated protein kinase), and Akt/PKB (protein kinase B) can regulate cyclin D1 protein expression in other cell types, pancreatic endocrine tumours were analysed with phospho-specific antibodies against the active forms of these proteins to elucidate a tIssue-specific regulatory mechanism of cyclin D1 in PETs. We found frequent activation of the p38/MAPK and Akt pathways, but down-regulation of the ERK pathway, in cyclin D1 overexpressing PETs. This study demonstrates that cyclin D1 overexpression is associated with human sporadic PET tumorigenesis, and suggests that this up-regulation may occur at the post-transcriptional level. These findings will direct future studies of PETs towards cell cycle dysregulation and the identification of key growth factor pathways involved in the formation of these tumours.

Topics: Adenoma, Islet Cell; Cell Differentiation; Cell Transformation, Neoplastic; Cyclin D1; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Pancreatic Neoplasms; Up-Regulation

Pancreatic ductal adenocarcinoma (PDAC) cell lines, MIA PaCa-2, and UK Pan-1, were used to investigate the role of ErbB2 in PDAC oncogenesis. Both these cell lines exhibit exogenous growth factor-independent proliferation that was attributed to the production of autocrine growth factors and/or overexpression of growth factor receptors. The exogenous growth factor-independent phenotype displayed by these PDAC cell lines was dependent on ErbB2 kinase activity since treatment of cells with tyrphostin AG879 prevented serum-free media (SFM) induction of cell proliferation. We determined that ErbB2 kinase contributed to aberrant cell cycle regulation in PDAC through the induction of cyclin D1 levels and the suppression of p21(Cip1) and p27(Kip1). Inhibition of ErbB2 kinase led to cell cycle arrest marked by an increased association of p27(Kip1) with cdk2 and reduced levels of phosphorylated pRb. We further observed constitutive STAT3 activation in the PDAC cell lines and an increase in STAT3 activation upon stimulating quiescent cells with SFM. Inhibitors of ErbB2 kinase blocked STAT3 activation, whereas inhibition of EGFR kinase led to a slight reduction of STAT3 activation. STAT3 was coimmunoprecipitated with ErbB2. SFM stimulation caused an increase in the association of ErbB2 and STAT3, which was blocked by inhibition of ErbB2 kinase. Expression of a STAT3 dominant negative prevented SFM-stimulated cell proliferation of MIA PaCa-2 cells, suggesting that activation of STAT3 by ErbB2 is required for a growth factor-independent phenotype of these cells. Consistent with this observation in PDAC cell lines, we found that most PDAC tumor specimens (10 of 11) showed constitutive activation of STAT3 and that ErbB2 was readily detected in most of these tumors (nine of 11). We believe that these findings indicate a novel mechanism of oncogenesis in PDAC and may suggest future therapeutic strategies in the treatment of PDAC.

Topics: CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; ErbB Receptors; Growth Substances; Humans; Pancreatic Neoplasms; Phosphorylation; Receptor, ErbB-2; Retinoblastoma Protein; STAT3 Transcription Factor; Trans-Activators; Tumor Suppressor Proteins; Tyrosine

The consumption of green tea is associated with a lower risk of several types of human carcinomas. A number of studies have focused on the possible mechanisms of cancer prevention by tea extracts, especially polyphenols such as epigallocatechin-3-gallate (EGCG).. Green tea-derived EGCG was tested in human pancreatic carcinoma cells. The cells (PANC-1, MIA PaCa-2, and BxPC-3) were treated with different doses of EGCG (0, 25, 50, 100, and 200 micromol/L) for 48 hours in culture medium. Proliferation of pancreatic carcinoma cells was measured by means of the WST-1 colorimetric assay. For the study of cell invasion, the cells were incubated with 100 micromol/L EGCG for 2 hours. Then, the cells were added into the cell insert, coated with Matrigel basement membrane matrix. After incubation at 37 degrees C for 24 hours, the cells that had invaded through the Matrigel were counted visually under the microscope.. The growth of all three pancreatic carcinoma cells was significantly suppressed by EGCG treatment in a dose-dependent manner. EGCG treatment caused significant suppression of the invasive ability of pancreatic carcinoma cells PANC-1, MIA PaCa-2, and BxPC-3 but did not affect the cell cycle protein cyclin D1.. EGCG may be a potent biologic inhibitor of human pancreatic carcinomas, reducing their proliferative and invasive activities.

Topics: Antineoplastic Agents, Phytogenic; Biocompatible Materials; Carcinoma; Catechin; Cell Division; Collagen; Cyclin D1; Drug Combinations; Humans; Laminin; Neoplasm Invasiveness; Pancreatic Neoplasms; Proteoglycans; Tea; Tumor Cells, Cultured

Topics: Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Pancreatic Neoplasms; Precancerous Conditions; Tumor Suppressor Protein p53

Prognostic indicators in pancreatic cancer (PC) are poorly defined and difficult to quantify preoperatively, hence they may lead to inappropriate patient selection for treatment. We examined the protein expression of key cell-cycle regulatory and cell-signaling molecules that occur at high frequency in PC and assessed their relationship to clinicopathologic parameters, response to operative resection, and outcome.. We identified 348 patients with pancreatic ductal adenocarcinoma and assessed the influence of reported clinicopathologic prognostic factors and the expression of the cell-cycle regulatory genes p21(WAF1/CIP1) (CDKN1A), cyclin D1 (CCND1), p53, and p16(INK4A) (CDKN2) and the cell-signaling molecule DPC4/Smad4 (MADH4) using immunohistochemistry in a subgroup of 129 patients.. Independent prognostic factors in resected patients were tumor size greater than 45 mm (P =.0015), involvement of surgical margins (P <.0001), and perineural invasion (P =.014). Loss of DPC4/Smad4 expression cosegregated with resectability (P <.0001) and was associated with improved survival after resection (P <.0001), whereas resection did not improve survival in patients whose tumor expressed DPC4/Smad4 (P =.5). Aberrant expression of p21(WAF1/CIP1), cyclin D1, p53, or p16(INK4A) was not associated with a difference in survival.. Tumor size (> 45 mm), resection margin involvement, and perineural invasion were independent prognostic factors. Preoperative assessment of DPC4/Smad4 expression has potential as a prognostic indicator in patients with PC since resection did not benefit those patients whose cancers expressed DPC4/Smad4 and accurate assessment of DPC4/Smad4 expression, unlike tumor size, margin status, and perineural invasion, does not require resection.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Down-Regulation; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Smad4 Protein; Survival Analysis; Trans-Activators; Treatment Outcome; Tumor Suppressor Protein p53; Up-Regulation

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in certain human cancers; ligand-induced PPARgamma activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARgamma ligands is not entirely known.. The purpose of this study was to examine the effect of PPARgamma ligands on pancreatic cancer cell growth and invasiveness.. The effect of two PPARgamma ligands, 15 deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ2 on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined.. Both 15d-PGJ2 and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ2 resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21waf1 and p27kip1. In addition, 15d-PGJ2 treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ2, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity.. These results demonstrate that PPARgamma ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.

Topics: 3T3 Cells; Adenocarcinoma; alpha Catenin; Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cadherins; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Division; Collagen; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cyclooxygenase 2; Cytoskeletal Proteins; Drug Combinations; Enzyme Induction; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Laminin; Ligands; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Proteoglycans; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

Peroxisome proliferator-activated receptor gamma (PPARgamma) forms a heterodimeric DNA-binding complex with the retinoid X receptor (RXR) and regulates the transcription of its target genes. Activation of PPARgamma has been shown to induce G1 arrest and to inhibit cell growth of human pancreatic carcinoma cell lines. The purpose of the present study was to examine the effect of ligand activation of PPARgamma and RXR on cell growth and on the expression of G1 cyclins in a pancreatic cancer cell line PANC-1, which expresses PPARgamma at high levels. Troglitazone, a specific ligand for PPARgamma, was found to cause a reduction in the growth rate and induced G1 cell cycle arrest and this effect was additive with that of 9-cis retinoic acid (9-cis RA), a ligand for RXR. Of the G1 cyclins tested, troglitazone specifically reduced the expression of cyclin D1 mRNA and the corresponding protein and this effect was also additive with 9-cis RA. These results suggest that the activation of PPARgamma together with RXR may be useful for the suppression of pancreatic cancer cell growth through the reduction in cyclin D1 levels.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Carcinoma; Cell Division; Chromans; Cyclin D1; Dose-Response Relationship, Drug; Drug Synergism; G1 Phase; Humans; Pancreatic Neoplasms; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Transfection; Tretinoin; Troglitazone; Tumor Cells, Cultured

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.

Topics: Apoptosis; Calpain; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Survival; Colonic Neoplasms; Cyclin B; Cyclin B1; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Cysteine Endopeptidases; DNA Fragmentation; Flow Cytometry; G1 Phase; Giant Cells; Humans; Keratins; Lysosomes; Microscopy, Electron; Models, Biological; Multienzyme Complexes; Osmosis; Osmotic Pressure; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; S Phase; Time Factors; Tumor Cells, Cultured

Intraductal papillary mucinous tumours (IPMT) of the pancreas constitute a unique pathological entity with an overall incidence of associated invasive malignancy of 20%. The malignant potential of an individual IPMT cannot be accurately predicted. Preoperative estimation of the risk of associated invasive malignancy with IPMT would be of significant clinical benefit. As aberrations in cell cycle regulatory genes are associated with the progression of precursor pancreatic ductal lesions to invasive adenocarcinoma, we examined expression of key cell cycle regulatory genes in the cyclin D1/retinoblastoma pathway and the transforming growth factor beta/Smad4 signalling pathway in a cohort of patients with surgically resected IPMT.. Sections of formalin fixed paraffin embedded pancreatic tissue from a cohort of 18 patients with IPMT were examined using immunohistochemistry for protein expression of cell cycle regulatory genes p16(INK4A), p21(CIP1), p27(KIP1), cyclin D1, pRb, and p53, as well as the cell signalling molecule Smad4. A comparison of expression levels was made between adenoma/borderline IPMT (10 patients) and intraductal papillary mucinous carcinoma (IPMC) (eight patients, four of whom harboured invasive carcinoma). Statistical analysis was performed using the chi(2) and Fisher's exact tests.. Aberrant expression of the proteins examined increased in frequency from adenoma/borderline IPMT to IPMC. Specifically, there was a significantly greater incidence of loss of p16(INK4A) expression in IPMC: 8/8 lesions (100%) compared with 1/10 (10%) adenoma/borderline IPMT (p<0.001). Similarly, loss of Smad4 expression was associated with IPMC: 3/8 (38%) versus adenoma/borderline IPMT 0/10 (p<0.03). Loss of Smad4 expression within the IPMT was the best marker for the presence of invasive carcinoma (p<0.001).. These data indicate that loss of p16(INK4A) and Smad4 expression occur more frequently in IPMC alone, or with associated invasive carcinoma, compared with adenoma/borderline IPMT. Aberrant protein expression of these cell cycle regulatory genes in IPMT and pancreatic intraepithelial neoplasia in the current model of pancreatic cancer progression suggest similarities in their development and may also represent the subsequent risk of invasive carcinoma.

Topics: Adenocarcinoma, Mucinous; Aged; Aged, 80 and over; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Retinoblastoma Protein; Smad4 Protein; Trans-Activators; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

Topics: Animals; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Disease Models, Animal; Disease Progression; Female; G1 Phase; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype; Proto-Oncogene Proteins; ras Proteins; S Phase; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Solid pseudopapillary tumor of the pancreas was studied in a 20-year-old woman and a 54-year-old woman. In the younger patient, the tumor had metastasized to the liver 8 years after distal pancreatectomy. In both neoplasms, the distinct histologic pattern of solid, pseudopapillary, and degenerative cystic areas was present. Analysis by means of immunohistochemistry revealed a diffuse expression for vimentin, neuron-specific enolase, and a focal positivity for al-antitrypsin, whereas epithelial markers were negative in the tumor of the older patient and only focally expressed in the tumor of the younger patient. Immunohistochemical analysis of cell cycle-associated proteins provided an overexpression of cyclin D1 and cyclin D3 in both tumors, although to varying degrees. In addition, the cyclin-dependent kinase inhibitors p21, and to a lesser extent p27, were up-regulated just as mdm2. There was no accumulation of p53 protein, and Ki67-positive cells were extremely scarce. Analysis of the liver metastases showed an immunoreactive profile similar to that of the primary tumor. The results show a deregulation of the cell cycle with overexpression of cell cycle-activating proteins D1 and D3 and a probably counterbalancing upregulation of the cyclin-dependent kinase inhibitors p21 and p27. The findings may explain the low pool of Ki67-reactive tumor cells and the generally good clinical outcome of these tumors. Whether a more profound dysbalance of the cell cycle regulation is responsible for the development of metastatic disease remains to be clarified.

Topics: Biomarkers, Tumor; Carcinoma, Papillary; Child; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Female; Humans; Microfilament Proteins; Middle Aged; Muscle Proteins; Pancreatic Neoplasms; Up-Regulation

Beta-catenin is a component of the E-cadherin-catenin cell adhesion complex. It plays also a role in intracellular signaling and can function as an oncogene when it binds to the T-cell factor 4 (Tcf4)-binding site in the promoter region of cyclin D1 and transactivates genes after translocation to the nucleus. We evaluated the immunohistochemical expression pattern of beta-catenin in relationship with cyclin D1 overexpression, tumor grade, clinicopathologic parameters and patients' survival in 43 ductal adenocarcinomas of the pancreas and 5 normal pancreatic tissues. We were able to show that, both reduced membranous beta-catenin expression (25 of 43, 58.1%) and accumulation of beta-catenin in the cytoplasm (28 of 43, 65.1%) correlated significantly with cyclin D1 overexpression (both p < 0.0005). Furthermore, we could show a clear correlation between reduced membranous expression and ectopic cytoplasmic expression of beta-catenin (p < 0.0005). Among patients with carcinomas showing no cytoplasmic expression, the 1-year survival was 86.6% whereas among patients with carcinomas showing cytoplasmic expression only 35.7% survived 1 year (p < 0.01). Co-precipitation experiments revealed reduced beta-catenin bound to the E-cadherin-catenin complex in pancreatic tumor tissues compared with normal pancreatic tissues. These results suggest that beta-catenin may be involved in the tumorigenesis of pancreatic cancer and exhibited its effects mainly by the transactivation of cyclin D1.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Biomarkers, Tumor; Blotting, Western; Cell Membrane; Cyclin D1; Cytoplasm; Cytoskeletal Proteins; Female; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Precipitin Tests; Prognosis; Survival Analysis; Trans-Activators

We recently showed that cyclin D1 is overexpressed in human pancreatic carcinoma cells, and that this overexpression correlates significantly with a poor prognosis.. To assess the interrelations of epidermal growth factor (EGF), EGF receptor (EGFR), and cyclin D1 in human pancreatic carcinoma.. In pancreatic carcinoma cell lines (BxPC-3, AsPC-1), cell cycle analysis revealed an increase in cells in the S/G1 phase between 18 and 30 hours after stimulation with 50 ng/mL EGF. Cyclin D1 mRNA increased after 2 hours, corresponding to an increase in cyclin D1 protein, with the maximum level between 7.5 and 10 hours after stimulation, as demonstrated by Western blot analysis. We performed immunohistochemical analysis on 61 adenocarcinoma tissues for the expression of EGF, EGFR, and cyclin D1 and demonstrated an overexpression in the tumor cells in 51%, 54%, and 62.3%, respectively, whereas normal human pancreas stained negative for all of the three factors. Interestingly, EGF and EGFR expression correlated significantly with the cyclin D1 expression in human pancreatic tumor cells (p < 0.001 and p < 0.01, respectively).. These results demonstrate that cyclin D1 overexpression in the tumor cells of pancreatic carcinoma tissue is at least partly dependent on the mitogenic effects of EGF signaling through the EGFR.

Topics: Blotting, Western; Cell Cycle; Cell Division; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; G1 Phase; Gene Expression; Humans; Immunohistochemistry; Kinetics; Pancreatic Neoplasms; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S Phase; Tumor Cells, Cultured

Pancreatic cancer (PC) is thought to develop through a series of duct lesions termed pancreatic intraepithelial neoplasia (PanIN). Characterization of the molecular pathology of these lesions may lead to additional understanding of pancreatic ductal carcinogenesis. We examined the protein expression of four functionally related genes, p21(WAF1/CIP1) (CDKN1A), p53, cyclin D1 (CCND1), and DPC4/Smad4 (MADH4), aberrations of which are associated with PC, within 451 PanIN lesions present in the pancreata of 60 patients. p21(WAF1/CIP1) overexpression was present in the normal ducts of 9% of patients and increased progressively to 16% of patients with PanIN-1A lesions, to 32% of patients with PanIN-1B lesions, 56% of patients with PanIN-2 lesions, 80% of patients with PanIN-3 lesions, and 85% of patients with invasive carcinomas (P < 0.01). p53 and cyclin D1 overexpression occurred predominantly in PanIN-3 lesions (P < 0.01), and loss of DPC4/Smad4 expression occurred predominantly in PanIN-3 lesions and invasive carcinoma (P < 0.01). In addition, p21(WAF1/CIP1) overexpression occurred independently of p53 and DPC4/Smad4 expression within invasive carcinoma and PanIN-3 lesions. Cyclin D1 overexpression or loss of DPC4/Smad4 expression was apparent in 85% of invasive carcinomas but in only 14% of PanIN-2 lesions. These data demonstrate that overexpression of p21(WAF1/CIP1) occurs early in the development of PanIN, before aberrations in p53, cyclin D1, and DPC4/Smad4 expression. p21(WAF1/CIP1) overexpression, independent of p53 and/or DPC4/Smad4 expression, may reflect increased Ras activity, either directly through activating K-ras mutations or as a consequence of HER-2/neu (ERBB2) overexpression, both of which are common in PC and in early events in the development of PanIN. These data support further the current progression model for PC and demonstrate that aberrant expression of key cell cycle regulatory genes may be important in the early development and progression of PanIN.

Topics: Carcinoma, Pancreatic Ductal; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Progression; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Precancerous Conditions; Smad4 Protein; Trans-Activators; Tumor Suppressor Protein p53

The molecular pathogenesis of human pancreatic endocrine tumors (PETs) is poorly understood. Three independent animal models have pointed to the pivotal role of the G1/S cell cycle transition in pancreatic endocrine cell proliferation. We thus hypothesized that the cell cycle regulator cyclin D1 may contribute to the pathogenesis of human PETs. Overexpression of cyclin D1 was identified in 43% of cases, and no correlation was observed with clinical phenotype. The novel observation of frequent overexpression of cyclin D1 suggests that this established oncogene may be implicated in the pathogenesis of human PETs. The absence of detectable alterations in cyclin D1 genomic structure suggests that the mechanism for its oncogenic activation in PETs may be transcriptional or posttranscriptional.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Cyclin D1; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multiple Endocrine Neoplasia; Multiple Endocrine Neoplasia Type 1; Pancreatic Neoplasms

Previous studies of molecular prognostic markers following resection for exocrine pancreatic cancer have produced conflicting results. Our aim was to undertake a comprehensive analysis of potentially useful molecular markers in a large, multicentre patient population and to compare these markers with standard pathological prognostic variables. Formalin-fixed, paraffin-embedded specimens of pancreatic ductal adenocarcinoma were analysed from 157 patients [100 men and 57 women with a median (range) age of 60 (33-77) years] who had undergone pancreatectomy. Immunohistochemistry was used to detect expression of p16(INK4), p53, p21(WAF1), cyclin D1, erbB-2 and erbB-3. Mutations in codons 12 and 13 of the K-ras oncogene were detected by SSCP and sequencing following DNA extraction and amplification by PCR. The median (range) survival post-resection was 12.5 (3-83) months. Abnormalities of p16(INK4), p53, p21(WAF1), cyclin D1, erbB-2 and erbB-3 expression were found in 87%, 41%, 75%, 72%, 33% and 57% of cases, respectively. There was no significant correlation between expression of any of these markers and patient survival. K-ras mutations were found in 73 (75%) of 97 cases with amplifiable DNA. The presence of K-ras mutation alone did not correlate with survival, but there were significant differences in survival according to the type of K-ras mutation (p = 0.0007). Reduced survival was found in patients with GaT, cGT and GcT K-ras mutations compared to GtT, aGT and GaC mutations. In conclusion, survival was associated with type of K-ras mutation but not expression of p16(INK4), p53, p21(WAF1), cyclin D1, erbB-2 and erbB-3.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Mutational Analysis; Female; Follow-Up Studies; Genes, erbB; Genes, erbB-2; Genes, ras; Humans; Immunohistochemistry; Male; Middle Aged; Mutation; Pancreatic Neoplasms; Polymorphism, Single-Stranded Conformational; Prognosis; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Tumor Suppressor Protein p53

In the present study, we show that 2-(2-hydroxyethylsulfaryl)-3-methyl-1,4-naphthoquinone, or CPD 5, is a potent growth inhibitor for pancreas cancer cell lines (ID(50): 21.4 +/- 3.8, 31.8 +/- 2.7 and 55.2 +/- 4.5 microM for MiaPaCa, Panc-1 and BxPc3, respectively). It induced protein tyrosine phosphor-ylation of hepatocyte growth factor (HGF) receptor (c-Met) or epidermal growth factor receptor (EGFR), which increased progressively to a maximum level at 30 min in Panc-1 cells. The receptor phosphorylation by CPD 5 was indicated to be functional, since these receptors were found to bind with Grb2 or SOS1 protein. CPD 5 was also suggested to induce phosphorylation of external signal-regulated kinase (ERK). EGF induced cell proliferation through ERK phosphorylation, since U0126, which is an inhibitor of ERK phosphorylation, abrogated the increase of cyclin D1 by EGF. HGF increased the amount of p27 protein, suggesting that it is associated with cell differentiation. By contrast, U0126 reduced CPD 5-induced cell death. On two-dimensional electrophoresis, we found an extra type of phospho-ERK, and this was completely and selectively abolished by U0126. These results suggest that ERK phosphorylation, especially the extra spot on two-dimensional gel, is critically associated with CPD 5-mediated cell death.

Topics: Antineoplastic Agents; Cell Death; Cell Division; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Microfilament Proteins; Mitogen-Activated Protein Kinases; Muscle Proteins; Naphthoquinones; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-met; Tumor Cells, Cultured; Vitamin K

Investigate the effects of blocking the TGF alpha-EGFR autocrine loop on the growth of human pancreatic carcinoma.. The pCMV-AS-EGFR, a recombinant vector expressing antisense EGFR under the control of human CMV promotor was constructed and transfected the transformant PC-7/AS-TGF alpha cell line cells, which had been transfected by a recombinant retroviral vector expressing antisense TGF alpha. G418 resistant colonies were isolated and identified as PC-7/AS-TGF alpha/AS-EGFR. The integration and expression of exogenous and endogenous genes were detected by Southern blot and Northern blot analysis. Apoptosis was detected by DNA fragmentation, flow cytometry and in situ cell death detection.. The cells which were double-transfected by the recombinant vectors showed the integration and expression of exogenous genes, and the downregulation of endogenous EGFR and cyclin D1 mRNA. 125I-EGF ligand binding test showed the binding affinity of the EGFR on the cell surface also reduced. The inhibition effect of cotransfecting of antisense TGF alpha and antisense EGFR was remarkable as compared with that of antisense TGF alpha alone. The incorporation rate of 3H-TdR reduced from 25% to 14.5%, and the growth inhibition rate increased from 78.6% to 86.0%. The ability of soft agar colony-formation was completely suppressed.. These observations strongly support that the blockage of the expression of autocrine growth factor TGF alpha and its receptor EGFR was a potent way in circumventing the malignant properties of the pancreatic carcinoma cells.

Topics: Cell Division; Cell Line, Transformed; Cyclin D1; ErbB Receptors; Humans; Pancreatic Neoplasms; RNA, Antisense; RNA, Messenger; Transfection; Transformation, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. Inhibition of cyclin D1 expression has been recently shown (M. Kornmann, et al., J. Clin. Invest, 101: 344-352, 1998) to suppress pancreatic cancer cell growth and increase cytotoxic actions of cisplatinum. The aim of the present study was to determine whether inhibition of cyclin D1 expression also modulates the effects of other antineoplastic drugs and whether it is associated with alterations in the level of expression of drug resistance genes. The suppression of cyclin D1 expression after the stable transfection of a cyclin D1 antisense construct in PANC-1 and COLO-357 human pancreatic cancer cells resulted in a significant increase in sensitivity to the fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine and to mitoxantrone. All of the antisense-expressing dones exhibited a decrease in thymidylate synthase and an increase in thymidine phosphorylase mRNA expression as determined by reverse transcription-PCR analysis and decreased levels of MDR-1 and MRP mRNA as determined by Northern blotting. These findings demonstrate that the inhibition of cyclin D1, in addition to suppressing the growth of pancreatic cancer cells, enhances their responsiveness to multiple chemotherapeutic agents and suggest that this effect may be due to the altered expression of several chemoresistance genes.

Topics: Adenocarcinoma; Cyclin D1; Drug Resistance, Neoplasm; Enzyme Induction; Floxuridine; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Mitoxantrone; Neoplasm Proteins; Oligonucleotides, Antisense; Pancreatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Thymidine Phosphorylase; Thymidylate Synthase; Transfection; Tumor Cells, Cultured

The FRAP-p70s6K signaling pathway was found to be constitutively phosphorylated/active in MiaPaCa-2 and Panc-1 human pancreatic cancer cells and a pancreatic cancer tissue sample as judged by the retarded electrophoretic mobility of the two major FRAP downstream targets, p70s6K and 4E-BP1. Treatment of cells with rapamycin, a selective FRAP Inhibitor, inhibited basal p70s6K kinase activity and induced dephosphorylation of p70s6K and 4E-BP1. Moreover, rapamycin inhibited DNA synthesis as well as anchorage-dependent and -independent proliferation in MiaPaCa-2 and Panc-1 cells. Finally, rapamycin strikingly inhibited cyclin D1 expression in pancreatic cancer cells. Thus, inhibitors of the constitutively active FRAP-p70s6K pathway may provide a novel therapeutic approach for pancreatic cancer.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Cell Cycle; Cell Cycle Proteins; Culture Media, Serum-Free; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Humans; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Neoplasm Proteins; Pancreatic Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Protein Processing, Post-Translational; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Suppressor Proteins

Transforming growth factor-beta1 (TGF-beta1) inhibits cell growth in susceptible cells by interacting with a family of protein kinases that control cell cycle progression. In the present study, we investigated the effects of TGF-beta1 on cyclin D1 expression and activity in COLO-357 human pancreatic cancer cells. TGF-beta1 increased cyclin D1 mRNA and protein levels. Nuclear runoff transcription and protein synthesis inhibition by cycloheximide revealed that this increase was, in part, due to increased cyclin D1 mRNA synthesis. Despite its stimulatory effects on cyclin D1 levels, TGF-beta1 inhibited cyclin D1-associated kinase activity and the growth of COLO-357 cells. Furthermore, suppression of cyclin D1 expression with a cyclin D1 antisense cDNA resulted in loss of TGF-beta1-mediated growth inhibition in association with reduced induction of cyclin D1, p21(C)(ip)(1) and plasminogen activator inhibitor-1 (PAI-1). Concomitantly, there was a marked decrease in the levels of the type I TGF-beta receptor (TbetaRI). Our findings suggest that in some cell types cyclin D1 expression may be important for TGF-beta1-mediated signaling and that cyclin D1 may be involved in the transcriptional regulation of TbetaRI.

Topics: Activin Receptors, Type I; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Down-Regulation; Growth Inhibitors; Humans; Insulin-Like Growth Factor I; Oligonucleotides, Antisense; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. Recent studies have demonstrated that elevated cyclin D1 levels correlate with decreased survival in human pancreatic cancer. In this study we expressed in a stable manner a cyclin D1 antisense cDNA construct in PANC-1 human pancreatic cancer cells. Expression of the antisense construct caused a decrease in cyclin D1 mRNA and protein levels and in cyclin D1-associated kinase activity. Antisense expressing clones displayed significantly increased doubling times, decreased anchorage-dependent and -independent basal growth, and complete loss of tumorigenicity in nude mice. EGF, FGF-2, and IGF-I enhanced mitogen-activated protein kinase activity in antisense expressing clones, but failed to stimulate their proliferation. In contrast, all three growth factors were mitogenic in parental cells. Furthermore, the inhibitory effect of cisplatinum on cell proliferation was enhanced markedly in the antisense expressing clones. These findings indicate that cyclin D1 overexpression contributes to abnormal growth and tumorigenicity in human pancreatic cancer and to the resistance of pancreatic cancer to chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Antisense Elements (Genetics); Cisplatin; Cyclin D1; DNA, Complementary; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Transfection; Tumor Cells, Cultured

We investigated the expression of interleukin 1beta-converting enzyme (ICE; caspase-1) in human adenocarcinomas of the pancreas. Immunohistochemistry and Western blot analyses revealed an overexpression of ICE in 71 and 80% of tumor cells, respectively. Also, on a mRNA level, ICE mRNA was overexpressed in 45% of the cases, as compared to normal pancreatic tissue. Interestingly, the overexpression of ICE in tumor cells correlated significantly with the overexpression of cyclin D1, epidermal growth factor, and epidermal growth factor receptor (P < 0.0005, P < 0.05, and P < 0.002, respectively), which are involved in cell cycle progression and proliferation in human pancreatic carcinoma. This is the first report concerning ICE expression in human carcinomas; however, the exact mechanism underlying these close correlations warrant further research.

Topics: Adenocarcinoma; Adult; Aged; Blotting, Western; Caspase 1; Cyclin D1; Cysteine Endopeptidases; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Pancreas; Pancreatic Neoplasms; RNA, Messenger

To observe the effects of antisense (AS) cyclin D1 expressing vector on the cell growth and apoptosis of pancreatic carcinoma.. Examination of the amplification and expression of cyclin D1 in 5 human pancreatic carcinoma cell lines. Our study found the gene amplification and overexpression of cyclin D1 in PC-7 cell line cells. We then constructed the antisense cyclin D1 vector and transfected the PC-7 cell line with lipofectin. The resultant transformant cell line, PC-7/AS-cyclin D1, showed the expression of exogenous antisense cyclin D1 mRNA and down regulation of endogenous cyclin D1 mRNA expression and inhibition of its protein synthesis detected by Northern blot and Western blot respectively.. The transformant cells showed retardation of cell growth and partial reversion of the malignant phenotype, including decrease of the rates of cell growth, DNA synthesis, cell proliferation and metabolism, and also the ability of soft agar colony-formation. The tumorogenesis of the transformant cells in nude mice was suppressed. G1 arrest was revealed by flow cytometry. Apoptosis was identified by DNA fragmentation and in situ TUNEL detection.. Down scaling of the expression of cyclin D1, which plays an important role in the regulation of the cell cycle, can effectively inhibit the proliferation of carcinoma cells and increase cell apoptosis.

Topics: Apoptosis; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Gene Amplification; Genes, bcl-1; Humans; Pancreatic Neoplasms; RNA, Antisense; Transfection; Tumor Cells, Cultured

We have investigated the expression of cyclin D1 in adenocarcinoma of the pancreas and the relevance of cyclin D1 expression to clinical outcome. In comparison to normal pancreas, Southern blot analyses revealed amplification of the cyclin D1 coding gene in 25% of the cases, whereas with reverse transcription-PCR, overexpression of mRNA was observed in 82% of the examined tissues. Immunohistochemically, we could demonstrate nuclear overexpression in tumor cells in 68.4%, and this protein accumulation correlated significantly with poor prognosis [median survival, 18.1 versus 10.5 months; P < 0.01 (chi2 test)].

Topics: Adult; Aged; Carcinoma; Cyclin D1; Cyclins; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Oncogene Proteins; Pancreatic Neoplasms; Prognosis; RNA, Messenger; RNA, Neoplasm; Survival Analysis

Somatic mutations in the retinoblastoma-1 gene (RB1) and loss of RB1 protein function have been implicated in a number of human malignancies, but the role of RB1 gene and protein abnormalities in ductal pancreatic cancer (DPCA) is virtually unknown. We therefore analyzed expression of the RB1 protein immunohistochemically and/or by western blotting in a total of 54 sporadic and eight familial cases of archival and frozen DPCA and in 18 pancreatic carcinoma cell lines by using the antibodies RB-WL-1, 84-B3-1, and PMG3-245. Mutations in the RB1 promotor region and exons 13-21 of the RB1 gene were likewise examined by single-strand conformation polymorphism (SSCP) analyses and DNA sequencing of genomic DNA from 30 microdissected primary pancreatic tumors and the pancreatic carcinoma cell lines. Moreover, amplification and expression of a major regulatory component of RB1 function, cyclin D1, were assessed by southern and immunohistochemical analyses, respectively. The DPCAs were heterogeneous in both the intensity of RB1 nuclear staining and the percentage of immunoreactive cells. The tumors often had areas where RB1 staining was weak or absent adjacent to normal pancreatic tissue; however, only two of 32 archival cases and one of 30 frozen cases of DPCA completely lacked RB1 nuclear staining. Immunohistochemical and western blot analyses of 18 pancreatic carcinoma cell lines demonstrated the absence of RB1 expression in only two cell lines, Capan-1 and QGP-1. Analyses of the RB1 gene and promotor region by SSCP and DNA sequencing largely confirmed the immunochemical findings. Three of 30 primary carcinomas had abnormalities revealed by SSCP analyses. In one case a single base-pair deletion was confirmed in exon 18 and resulted in premature termination and the absence of detectable RB1 protein. A second case had TAC-->TTC missense mutation in exon 13. The third primary carcinoma could not be reliably sequenced because it had a low percentage of epithelial cells. The cyclin D1 gene was not amplified in any of the primary pancreatic tumors or cell lines examined. These immunochemical and molecular analyses of the RB1 tumor suppressor gene and cyclin D1 proto-oncogene in a large series of human pancreatic cancers and cell lines indicate that RB1 and cyclin D1 alterations occur during the development of some human DPCAs. Nevertheless, it is probable that alterations in cell-cycle regulation in DPCAs more frequently involve pathways other than those involving RB1

Topics: Base Sequence; Blotting, Southern; Blotting, Western; Carcinoma, Ductal, Breast; Cell Line; Cyclin D1; Cyclins; Genes, Retinoblastoma; Humans; Immunohistochemistry; Molecular Sequence Data; Oncogene Proteins; Pancreatic Neoplasms; Polymorphism, Single-Stranded Conformational; Proto-Oncogene Mas; Retinoblastoma Protein; Tumor Cells, Cultured