ag-490 and Pancreatic-Neoplasms

Drug-resistance is the key reason for the ineffectiveness of chemotherapy in pancreatic cancer. Thus, it is very important to explore the molecular mechanisms of drug‑resistance and the methods of effective intervention. In the present study, we investigated the effect of tyrphostin B42, also called AG490, on histone deacetylase (HDAC) inhibitor trichostatin A (TSA)‑induced resistance in pancreatic cancer cells (PCCs). Evidence from phase contrast microscope revealed that TSA‑resistant cells (PANC‑1‑TSA) had higher proliferative activity than non‑resistant cells (PANC‑1). This over‑proliferative activity induced by TSA may be associated with abnormal activation of JAK2/STAT3 signaling, which can be strengthened by interleukin‑6 (IL‑6), a STAT3‑upstream inducer, resulting in enhanced expression of STAT3-downstream target genes including c‑Myc, c‑Src, HIF‑1α, and CCND1. In addition, increased expression of Bcl‑2 mRNA and decreased expression of Bax mRNA in PANC‑1‑TSA cells indicated that TSA induced the inhibition of mitochondrial-dependent apoptosis in PCCs. Tyrphostin B42 treatment evidently antagonized the activation of IL‑6/JAK2/STAT3 in a dose‑dependent manner. As a result, tyrphostin B42 inhibited the over‑proliferative activity of PANC‑1‑TSA cells, and downregulated the expression of IL‑6/JAK2/STAT3‑downstream target genes. Moreover, tyrphostin B42 induced the apoptosis of PCCs by regulating the expression of mitochondrial‑related genes. Therefore, these findings demonstrated that tyrphostin B42 attenuated TSA‑mediated resistance in PCCs by antagonizing the IL‑6/JAK2/STAT3 signaling.

Topics: Apoptosis; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Interleukin-6; Janus Kinase 2; Pancreatic Neoplasms; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Pancreatic ductal adenocarcinoma (PDA) is a fatal and insidious malignant disease for which clinicians' tools are restricted by the current limits in knowledge of how tumor and stromal cells act during the disease. Among PDA hallmarks, neural remodeling (NR) and perineural invasion (PNI) drastically influence quality of life and patient survival. Indeed, NR and PNI are associated with neuropathic pain and metastasis, respectively, both of which impact clinicians' decisions and therapeutic options. The aim of this study was to determine the impact and clinical relevance of the peritumoral microenvironment, through pancreatitis-associated protein (PAP/REG3A) expression, on PNI in pancreatic cancer. First, we demonstrated that, in PDA, PAP/REG3A is produced by inflamed acinar cells from the peritumoral microenvironment and then enhances the migratory and invasive abilities of cancer cells. More specifically, using perineural ex vivo assays we revealed that PAP/REG3A favors PNI through activation of the JAK/STAT signaling pathway in cancer cells. Finally, we analyzed the level of PAP/REG3A in blood from healthy donors or patients with PDA from three independent cohorts. Patients with high levels of PAP/REG3A had overall shorter survival as well as poor surgical outcomes with reduced disease-free survival. Our study provides a rationale for using the PAP/REG3A level as a biomarker to improve pancreatic cancer prognosis. It also suggests that therapeutic targeting of PAP/REG3A activity in PDA could limit tumor cell aggressiveness and PNI.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Line; Cell Movement; Coculture Techniques; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lectins, C-Type; Mice; Microscopy, Fluorescence; Neoplasm Invasiveness; Nerve Fibers; Pancreatic Neoplasms; Pancreatitis-Associated Proteins; Perineum; Prognosis; Recombinant Proteins; Tyrphostins

Cancer cachexia syndrome is observed in 80% of patients with advanced-stage cancer, and it is one of the most frequent causes of death. Severe wasting accounts for more than 80% in patients with advanced pancreatic cancer. Here we wanted to define, by using an microarray approach and the Pdx1-cre;LSL-Kras(G12D) ;INK4a/arf(fl/fl) mice model, the pathways involved in muscle, liver, and white adipose tissue wasting. These mice, which develop systematically pancreatic cancer, successfully reproduced many human symptoms afflicted with this disease, and particularly cachexia. Using the profiling analysis of pancreatic cancer-dependent cachectic tissues we found that Jak2/Stat3 pathways, p53 and NFkB results activated. Thus, our interest was focused on the Jak2 pathways because it is pharmacologically targetable with low toxicity and FDA approved drugs are available. Therefore, Pdx1-cre;LSL-Kras(G12D) ;INK4a/arf(fl/fl) mice were treated with the Jak2 inhibitor AG490 compound daily starting at 7 weeks old and for a period of 3 weeks and animals were sacrificed at 10 weeks old. Body weight for control mice was 27.84 ± 2.14 g, for untreated Pdx1-cre;LSL-Kras(G12D) ;INK4a/arf(fl/fl) was 14.97 ± 1.99 g, whereas in animals treated with the AG490 compound the weight loss was significantly less to 24.53 ± 2.04 g. Treatment with AG490 compound was efficient since phosphorylation of Jak2 and circulating interleukin-6 (IL6) levels were significantly reduced in cachectic tissues and in mice respectively. In conclusion, we found that Jak2/Stat3-dependent intracellular pathway plays an essential role since its pharmacological inhibition strongly attenuates cachexia progression in a lethal transgenic pancreatic cancer model.

Topics: Adenocarcinoma; Adipose Tissue, White; Animals; Body Weight; Cachexia; Gene Expression Profiling; Interleukin-6; Janus Kinase 2; Liver; Mice; Mice, Transgenic; Muscle, Skeletal; Pancreatic Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; STAT3 Transcription Factor; Time Factors; Tyrphostins

Signal transducer and activator of transcription 3 (Stat3) is a member of the Janus-activated kinase(Jak)/Stat signaling pathway. Abnormal activation of Stat3 plays a critical role in metastasis and invasion in varieties of human tumors including pancreatic cancer. This study aimed to investigate the mechanisms of activation and blocking of the Stat3 signaling pathway and its effects on invasion and metastasis of human pancreatic cancer cells.. The Jak inhibitor AG490 and interleukin-6 (IL-6) were added to the culture media of human pancreatic cancer cells SW1990 and Capan-2, respectively. Cell growth was measured by MTT assays. Western blotting and immunocytochemistry were performed to detect phosphorylated Stat3 (p-Stat3) protein, while VEGF and MMP-2 mRNA and protein expression were examined with fluorescence quantitative polymerase chain reaction and Western blotting, respectively. The invasion ability of SW1990 and Capan-2 cells was determined by cell invasion assay.. Stat3 was activated by IL-6 in Capan-2 cells; protein expression of p-Stat3 was increased significantly in Capan-2 cells. IL-6 remarkably promoted the growth of Capan-2 cells (P < 0.05), and VEGF and MMP-2 mRNA and protein expression were increased significantly. Also, IL-6 increased the invasion ability of Capan-2 cells. AG490 inhibited Stat3 activation in SW1990 cells. Western blotting and immunocytochemistry analysis showed that p-Stat3 protein expression was decreased significantly with AG490 treatment in SW1990 cells. AG490 remarkably inhibited the growth of Capan-2 cells (P < 0.05), and VEGF and MMP-2 mRNA and protein expression was decreased significantly. And AG490 decreased the invasion ability of SW1990 cells.. Abnormal activation of Stat3 plays an important role in the invasion and metastasis of pancreatic cancer. Activation and blocking of the Stat3 signaling pathway can affect invasion ability and expression of the VEGF and MMP-2 genes in pancreatic cancer cells. The Stat3 signaling pathway may provide a novel therapeutic target for treatment of pancreatic cancer.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Interleukin-6; Matrix Metalloproteinase 2; Pancreatic Neoplasms; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Vascular Endothelial Growth Factor A

Mesothelin (MSLN) is a cell surface glycoprotein that is overexpressed in human pancreatic cancer. Although its value as a tumor marker for diagnosis and prognosis and as a preferred target of immunointervention has been evaluated, there is little information on the growth advantage of MSLN on tumor cells. In this study, we examined the effect of MSLN on pancreatic cancer cell proliferation, cell cycle progression, expression of cell cycle regulatory proteins, and signal transduction pathways in two pancreatic cancer cell lines, MIA-MSLN (overexpressing MSLN in MIA PaCa-2 cells) and BxPC-siMSLN (silencing MSLN in BxPC-3 cells). Increased cyclin E and cyclin-dependent kinase 2 expression found in MIA-MSLN cells correlated with significantly increased cell proliferation and faster cell cycle progression compared with control cells. BxPC-siMSLN cells showed slower proliferation and slower entry into the S phase than control cells. Signal transducer and activator of transcription protein 3 (Stat3) was constitutively activated in MIA-MSLN cells, but not in control cells. Inhibition of Stat3 activation in MIA-MSLN cells by the Janus-activated kinase-selective inhibitor tyrphostin AG490 was followed by a marked decrease in proliferation of the cells. Small interfering RNA against Stat3 significantly reduced the MIA-MSLN cell cycle progression with a concomitant decrease in cyclin E expression. Our data indicate that overexpression of MSLN in pancreatic cancer cells leads to constitutive activation of the transcription factor Stat3, which results in enhanced expression of cyclin E and cyclin E/cyclin-dependent kinase 2 complex formation as well as increased G(1)-S transition.

Topics: Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin E; Cyclin-Dependent Kinase 2; Enzyme Inhibitors; Gene Silencing; GPI-Linked Proteins; Humans; Janus Kinases; Membrane Glycoproteins; Mesothelin; Pancreatic Neoplasms; RNA, Small Interfering; S Phase; STAT3 Transcription Factor; Tyrphostins

Although dendritic cell (DC) function is impaired in pancreatic cancer patients, the underlying mechanisms are unknown. This study analyzed the soluble factors released by pancreatic cancer cells responsible for inhibiting DC differentiation and activation. Medium conditioned by a highly metastatic human pancreatic cancer cell line BxPC-3 [BxPC-3 conditioned medium (BxCM)] was mainly used for the study. Both CD34+ hematopoietic progenitor cell-derived and CD14+ monocyte-derived immature DCs and mature DCs (mDCs) were inhibited by BxCM. Allostimulation of CD4+ and CD8+ T cells by BxCM-treated mDCs was inefficient and resulted in production of lower levels of Th1 and Th2 cytokines. Antigen-specific T-cell activation capability was also reduced in BxCM-treated mDCs. Addition of exogenous interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), which were present in high amounts in BxCM, mimicked the inhibitory effect of BxCM on DC differentiation and maturation. IL-6 was able to suppress DC differentiation and G-CSF mainly acted on the suppressing allostimulatory capacity of DCs. In addition, pancreatic cancer patient sera were able to inhibit DC differentiation of CD14+ monocytes obtained from healthy donors. Depleting IL-6 or G-CSF from BxCM could reverse the DC-inhibitory properties of BxCM. Furthermore, BxCM, IL-6, or G-CSF led to the activation of signal transducer and activator of transcription 3 (STAT3) in CD14+ monocytes to different degrees. Blocking BxCM-induced STAT3 activation also reversed the inhibitory effect of BxCM on DC differentiation. Therefore, IL-6 and G-CSF in BxCM represent two main factors responsible for suppression of DC differentiation, maturation, and antigen presentation, and this suppression of DC functions may be due to the aberrant activation of STAT3 by BxCM.

Topics: Antigen Presentation; Antigens, CD34; Cell Differentiation; Cell Line, Tumor; Culture Media, Conditioned; Dendritic Cells; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Interleukin-6; Janus Kinase 2; Lipopolysaccharide Receptors; Lymphocyte Activation; Monocytes; Pancreatic Neoplasms; STAT3 Transcription Factor; T-Lymphocytes; Tyrphostins

Signal transducer and activator of transcription 3 (STAT3) is a central cytoplasmic transcription factor. Abnormal activation of STAT3 plays a critical role in oncogenesis and has been found frequently in a wide variety of human tumors including pancreatic cancer. In this study, we elucidated the significance of the STAT3 signaling pathway on metastatic potentials of pancreatic cancer. We found that phosphorylated STAT3 (p-STAT3) protein levels were significantly higher in highly metastatic SW1990 cells compared to the poorly metastatic CaPan-2 cells, which expressed weak levels of this protein. Furthermore, a Janus kinase (JAK) specific inhibitor, AG490, significantly inhibited the expression of p-STAT3, and subsequently reduced invasion and adhesion potential of SW1990 cells compared to the cells treated with vehicle only. Finally, inhibition of the STAT3 signaling pathway by AG490 also led to a decrease in matrix metalloproteinase-2 and vascular endothelial growth factor expression at the protein and mRNA levels. These results demonstrate that activation of the STAT3 signaling pathway plays an important role in the progression of pancreatic cancer and that inhibition of this pathway may be useful for an anti-invasive therapeutic option in pancreatic cancer.

Topics: Blotting, Western; Cell Adhesion; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Pancreatic Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Tyrphostins; Vascular Endothelial Growth Factor A

To investigate the effect and mechanism of blockade of STAT3 signaling pathway by JAK specific inhibitor-AG490 on invasion and metastasis of human highly metastatic pancreatic cancer line SW1990 in vitro.. AG490 was added into the culture media for SW1990 cells. The invasion ability of SW1990 cells was determined by cell invasion assay kit. Western blot was performed to detect the protein expression of the STAT3, phosphorylated STAT3 (p-STAT3), MMP-2 and VEGF. RT-PCR was performed to detect the mRNA expression of the MMP-2 and VEGF.. 20 micromol/L AG490 significantly inhibited the invasion ability of SW1990 cells and the inhibitory rate of invasion ability was (77.67 +/- 7.79) %. The use of AG490 not only markedly reduced the protein expression of p-STAT3, MMP-2 and VEGF, but also greatly reduced the mRNA expression of MMP-2 and VEGF.. Blocking STAT3 activation with AG490 can inhibit the invasion and metastasis ability of pancreatic cancer cells in vitro through down-regulation of MMP-2 and VEGF expression. Blocking STAT3 signaling pathway may provide a novel strategy in prevention of invasion and metastasis of pancreatic cancer.

Topics: Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Janus Kinases; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Vascular Endothelial Growth Factor A

1. Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K+ (KATP) channels was examined in the rat CRI-G1 insulinoma cell line. 2. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP; 3-5 mM), the leptin-induced hyperpolarization and increase in K+ conductance were completely inhibited. 3. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 microM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. 4. Application of the tyrosine kinase inhibitors genistein (10 microM), tyrphostin B42 (10 microM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 microM) in the pipette solution activated tolbutamide-sensitive KATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. 5. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 microM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 microM) prevented the actions of both leptin and tyrphostin B42. 6. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Androstadienes; Animals; Enzyme Inhibitors; Insulinoma; Leptin; Nitriles; Pancreatic Neoplasms; Phosphoric Monoester Hydrolases; Phosphorylation; Potassium Channels; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proteins; Rats; Tumor Cells, Cultured; Tyrosine; Tyrphostins; Wortmannin