Adenocarcinoma Cell Line MIA PaCa-2 Research Articles

Fc Receptor is Involved in Nk Cell Functional Anergy Induced by Miapaca2 Tumor Cell Line

ABSTRACT Impaired NK cytotoxicity has been linked to poor cancer prognosis, but its mechanisms are not clearly established. Increasing data demonstrate that NK cells lose cytotoxicity after interaction with NK cell-sensitive tumor cells. In this paper, we provide evidence that the human adenocarcinoma cell line MiaPaCa2 and TNFα and TGFβ-treated MiaPaCa2 cultures (MiaPaCa2-TT) induced functional anergy of NK cells via FGL2 protein. MiaPaCa2-TT cultures decreased expression of IFNγ, CD107a, DNAM-1, and stimulated expression of PD1 by NK cells, as well as inhibited their cytotoxic activity in a greater manner compared to the parental culture. More importantly, we found that co-cultivation with anergized NK cells decreased expression of IFNγ and CD107a by naïve NK cells, which supports the hypothesis of NK cell functional anergy transmission. The obtained results suggest a mechanism by which tumor cells may inhibit cytotoxic functions of tumor-infiltrating and circulating NK cells in cancer. Abbreviations: CFSE: Carboxyfluorescein diacetate succinimidyl ester; CSCs: Cancer stem cells; FGL2: Fibrinogen-like protein 2; mAbs: Monoclonal antibodies; MiaPaCa2: Human adenocarcinoma cell line; MiaPaCa2-ТТ: Adenocarcinoma cell line MiaPaCa2 cells stimulated with TNFα and TGFβ-1; PI: Propidium iodide; TGFβ: Transforming growth factor beta; TME: Tumor microenvironment; TNFα: Tumor necrosis factor alfa

Dynamic contrast-enhanced (DCE) MRI assessment of microvascular characteristics in the murine orthotopic pancreatic cancer model

ObjectThe aim of this study was to assess the dynamic contrast enhanced magnetic resonance imaging (DCE-MRI)-derived pharmacokinetic parameters between two contrast agents in a murine orthotopic pancreatic cancer model and to evaluate the tumor heterogenity and the potential association between kinetic parameters and angiogenic markers such as the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression by immunohistochemistry. Materials and methodsHuman pancreatic adenocarcinoma cell line MIAPaCa-2 was injected into the pancreas of BALB/C nu/nu mice. DCE-MRI was performed using Gd-DTPA and Gd-EOB-DTPA. Quantitative and semi-quantitative vascular parameters (Ktrans, Kep, Ve and AUC) were calculated by using a dedicated postprocessing software program. Values were compared with tumor rim, tumor core and the entire tumor. The MVD and VEGF expressions between tumor rim and tumor core were also compared. ResultsThere were no significant differences in Ktrans, Kep, Ve, and AUC values of the three groups when using Gd-DTPA. However there were significant differences in Ktrans, Kep, and AUC values of the three groups when using Gd-EOB-DTPA (P=0.014, 0.022, 0.007, respectively), in addition, the Ktrans and Kep values of tumor core were significantly lower than those of the entire tumor (adjusted P=0.014 and 0.027, respectively), the AUC values of core were significantly lower than those of the entire tumor and rim (adjusted P=0.039 and 0.009, respectively). Immunohistology results revealed that MVD and VEGF expression in the tumor rim was significantly higher than that in the core. There was positive correlation between AUC and MVD, VEGF. ConclusionThe murine orthotopic pancreatic cancer model provides an ideal animal model to study human pancreatic cancer. It can more sensitively semi-quantitatively and quantitatively analyze tumor angiogenesis through selecting the albumin-binding contrast agent.

Gallic Acid Is an Active Component for the Anticarcinogenic Action of Grape Seed Procyanidins in Pancreatic Cancer Cells

The aim of the present work was to evaluate the effects of a grape seed procyanidin extract (GSPE) on proliferation and apoptosis in the pancreatic adenocarcinoma cell line MIA PaCa-2 and identify the components of the extract with higher activity. The effects of the extract were analyzed on the proliferation and apoptosis processes in MIA PaCa-2 cells, as well as in the levels of the apoptosis markers Bcl-2 and Bax, the mitochondrial membrane potential, and reactive oxygen species levels. Finally, the components of the extract with higher effects were elucidated using enriched fractions of the extract and pure compounds. The results showed that GSPE inhibits cell proliferation and increases apoptosis in MIA PaCa-2 cells, which is primarily mediated by the downregulation of the antiapoptotic protein Bcl-2 and the depolarization of the mitochondrial membrane. GSPE also reduced the formation of reactive oxygen species. The component of the extract that possesses the highest antiproliferative and proapoptotic activity was gallic acid. In conclusion, GSPE acts as anticarcinogenic in MIA PaCa-2 cells, with gallic acid as the major single active constituent of the extract.

Extra- and Intracellular Imaging of Human Matrix Metalloprotease 11 (hMMP-11) with a Cell-penetrating FRET Substrate

Matrix metalloprotease 11 (MMP-11), a protease associated with invasion and aggressiveness of cancerous tissue, was postulated as a prognostic marker for pancreatic, breast, and colon cancer patients. Expression analysis, however, did not reveal localization and regulation of this protease. Thus, cellular tools for the visualization of MMP-11 are highly desirable to monitor presence and activity and to elucidate the functional role of MMP-11. Therefore, fluorescein-Dabcyl-labeled Foerster resonance energy transfer (FRET) substrates were developed. The design focused on enhanced peptide binding to human MMP-11, employing an unusual amino acid for the specificity pocket P1'. The addition of several arginines resulted in a cell-permeable FRET substrate SM-P124 (Ac-GRRRK(Dabcyl)-GGAANC(MeOBn)RMGG-fluorescein). In vitro evaluation of SM-P124 with human MMP-11 showed a 25-fold increase of affinity (k(cat)/K(m) = 9.16 × 10(3) m(-1) s(-1), K(m) = 8 μm) compared with previously published substrates. Incubation of pancreatic adenocarcinoma cell line MIA PaCa-2 and mamma adenocarcinoma cell line MCF-7 with the substrate SM-P124 (5 μm) indicated intra- and extracellular MMP-11 activity. A negative control cell line (Jurkat) showed no fluorescent signal either intra- or extracellularly. Negative control FRET substrate SM-P123 produced only insignificant extracellular fluorescence without any intracellular fluorescence. SM-P124 therefore enabled intra- and extracellular tracking of MMP-11-overexpressing cancers such as pancreatic and breast adenocarcinoma and might contribute to the understanding of the activation pathways leading to MMP-11-mediated invasive processes.

Probing the human kinome for kinases involved in pancreatic cancer cell survival and gemcitabine resistance

Except for gemcitabine, chemotherapeutic agents are ineffective with pancreatic adenocarcinoma because this cancer is resistant to apoptosis induction. Involvement of specific kinases in such resistance is likely. We developed a systematic strategy to screen the human kinome and select kinases whose inhibition in pancreatic cancer cells can increase 1) spontaneous apoptosis or 2) gemcitabine-induced apoptosis. The pancreatic adenocarcinoma cell line MiaPaCa-2 was transfected with 645 pairs of siRNAs directed to all human kinases. The same experiment was conducted in cells treated with 150 microM gemcitabine. Apoptosis was measured after 2 days and the results were normalized for cell viability. A panel of 56 kinases whose inhibition increased spontaneous apoptosis by at least 50% was established. Ten of them gave similar results on Panc1 and BxPC3 pancreatic adenocarcinoma cell lines. A panel of 83 kinases whose inhibition increased gemcitabine-induced apoptosis by 50% or more was also established. Twelve kinases appeared in both panels. A cumulative increase in apoptosis was observed when inhibiting simultaneously several kinases. Such a systematic approach allowed characterization of all kinases involved in pancreatic cancer cell survival and resistance to gemcitabine. Inhibitors of these kinases, used alone or in combination, might improve the treatment of pancreatic adenocarcinoma.

Melatonin Modulates Acid/Base Transport in Human Pancreatic Carcinoma Cells

Melatonin was found to improve pancreatic organfunction in diseased animals. To study whetherpancreatic bicarbonate secretion is stimulated bymelatonin, investigations were done in two humanductal pancreatic adenocarcinoma cell lines MIAPaCa-2 (MIA) and PANC-1 (PANC). Using thefluorescence pH-sensor BCECF-AM, we monitoredmelatonin effects on basal intracellular pH (pH_i), andon pH_i recovery after intracellular alkalinizationproduced by the removal of extracellular HCO₃^-/CO₂.Exposure to 1µM melatonin for 24hrs and presenceof the indoleamine during the experiment increasesthe basal pH_i. Moreover, pHi recovery and HCO₃^-secretion are facilitated after the alkaline load. Thesefindings are in line with the observed increase in mRNAexpression of the Na⁺/HCO₃^--cotransporter SLC4A4bfor the uptake and the Cl^-/HCO₃^--exchanger SLC26A6for the secretion of HCO₃^-. The reduction in Na⁺/H⁺-exchanger SLC9A1 mRNA would favor pH_i recoveryafter alkalinization, but it does not explain the initialincrease in pHi. This controversial effect and therequirement for continuous presence of melatoninthroughout the experiment suggest that nontranscriptionalsignalling may contribute to the effectsof melatonin on acid/base movements. In summary,we show a stimulatory effect of melatonin onbicarbonate secretion in the pancreatic cancer celllines which may help to prevent duodenal damage.

Epidermal growth factor mediates detachment from and invasion through collagen I and Matrigel in Capan-1 pancreatic cancer cells

BackgroundPancreatic adenocarcinoma is a highly invasive neoplasm. Epidermal growth factor (EGF) and its receptor are over expressed in pancreatic cancer, and expression correlates with invasion and metastasis. We hypothesized that EGF receptor and integrin signalling pathways interact in mediating cellular adhesion and invasion in pancreatic cancer, and that invasiveness correlates temporally with detachment from extracellular matrix.MethodsWe tested this hypothesis by investigating the role of EGF in mediating adhesion to and invasion through collagen I and Matrigel in the metastatic pancreatic adenocarcinoma cell line Capan-1. Adhesion and invasion were measured using in vitro assays of fluorescently-labeled cells. Adhesion and invasion assays were also performed in the primary pancreatic adenocarcinoma cell line MIA PaCa-2.ResultsEGF inhibited adhesion to collagen I and Matrigel in Capan-1 cells. The loss of adhesion was reversed by AG825, an inhibitor of erbB2 receptor signalling and by wortmannin, a PI3K inhibitor, but not by the protein synthesis inhibitor cycloheximide. EGF stimulated invasion through collagen I and Matrigel at concentrations and time courses similar to those mediating detachment from these extracellular matrix components. Adhesion to collagen I was different in MIA PaCa-2 cells, with no significant change elicited following EGF treatment, whereas treatment with the EGF family member heregulin-alpha elicited a marked increase in adhesion. Invasion through Matrigel in response to EGF, however, was similar to that observed in Capan-1 cells.ConclusionAn inverse relationship exists between adhesion and invasion capabilities in Capan-1 cells but not in MIA PaCa-2 cells. EGF receptor signalling involving the erbB2 and PI3K pathways plays a role in mediating these events in Capan-1 cells.

Thrombin enhances adhesion in pancreatic cancer in vitro through the activation of the thrombin receptor PAR 1

Background: Thrombin, the central enzyme of the coagulation cascade, induces proliferation in different solid tumours. The effect is mainly mediated through the functional thrombin receptor PAR 1, a member of the G-protein coupled receptor family. The aim of this study was to assess the role of thrombin on adhesion of pancreatic cancer to extracellular matrix proteins and endothelial cells in vitro. Materials and Methods: The human pancreatic adenocarcinoma cell line MIA PaCa-2 was treated with thrombin and the thrombin-receptor-activing peptide (TRAP), respectively. As a control the cells were pre-incubated with the thrombin-receptor-inhibiting peptide (T1). The cells were incubated on microtiter plates, which were pre-coated with extracellular matrix proteins (fibronectin, laminin, collagen IV) or human umbilical vein endothelial cells (HUVECs), for 30 and 60 min, respectively. The number of adherent cells were measured using the MTT method. ANOVA was used for statistical analysis. Results: Thrombin enhanced the adhesion of MIA PaCa-2 cells to extra-cellular matrix proteins and endothelial cells significantly (P≤0.001). The effects of thrombin could be mimicked by TRAP. Pre-incubation with T1 inhibited the effect. Conclusion: Thrombin enhances adhesion of pancreatic adenocarcinoma to extracellular matrix proteins and endothelial cellsin vitro. The effect is mediated through the thrombin receptor PAR 1. The results emphasize the role of thrombin and PAR 1 in pancreatic tumour biology.

Retroviral Transduction of Cancer Cell Lines with the Gene Encoding Drosophila melanogaster Multisubstrate Deoxyribonucleoside Kinase

Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors by combined gene/chemotherapy. We have recently cloned a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), and we have shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity of the enzyme, as well as its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present study, we evaluated Dm-dNK as a suicide gene by constructing a replication-deficient retroviral vector that expresses the enzyme. The human pancreatic adenocarcinoma cell line MIA PaCa-2 and a thymidine kinase-deficient osteosarcoma cell line were transduced with the recombinant virus. We showed that Dm-dNK can be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its C-terminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs, such as 1-beta-d-arabinofuranosylcytosine, 1-beta-d-arabinofuranosylthymine, (E)-5-(2-bromovinyl)-2'-deoxyuridine, 2-chloro-2'-deoxyadenosine, and 2',2'-difluorodeoxycytidine. These findings suggest that Dm-dNK may be used as a suicide gene in combined gene/chemotherapy of cancer.

Dehydroepiandrosterone-sulfate inhibits pancreatic carcinoma cell proliferation in vitro and in vivo

Background . Dehydroepiandrosterone-sulfate (DHEA-S) is a potent inhibitor of glucose-6 phosphate dehydrogenase, the rate limiting enzyme of the hexose monophosphate shunt, a biochemical pathway that provides substrate for DNA synthesis in neoplastic tissue. DHEA-S has been shown to inhibit the growth of neoplasms arriving from human skin, lung, colon, and mammary tissue. This study evaluates the effect of DHEA-S on human pancreatic cancer cell lines in vitro and in vivo. Methods . In vitro, the human pancreatic adenocarcinoma cell lines MiaPaCa-2, Capan-1, Capan-2, CAV and Panc-1 were treated with concentrations of 1.9 μmol/L to 1000 μmol/L DHEA-S in 1% dimethylsulfoxide (DMSO) for 5 consecutive days. Cell proliferation was determined by a nonradioactive cell proliferation assay and compared with DMSO treated controls. In vivo testing was performed by inoculating two cell lines, MiaPaCa-2 and Panc-1, into the flank of 40 male nude athymic mice in four study groups. After 1 week of growth, 667 mg/kg DHEA-S in 1% DMSO or 0.2 ml 1% DMSO alone in the control group was administered by daily intraperitoneal injection. Body weight and tumor size was recorded weekly, and tumor weight was measured after 3 weeks of treatment. Results . In vitro cell proliferation was decreased in the five cell lines by 36% to 62% of controls (p < 0.001) at 500 μmol/L DHEA-S. In vivo, after 2 weeks, tumor size was only 76% (p < 0.008) and 67% (p < 0.005) of the controls. After 3 weeks of treatment, tumor size was 73% (p < 0.001) and 53% (p < 0.001) of controls, and tumor weight was decreased by 73% in MiaPaCa-2 (p < 0.001) and 66% in Panc-1 (p < 0.001). Radioimmunoassay measurements of DHEA-S and testosterone from DHEA-S treated mouse plasma showed a significant increase in circulating levels of these hormones. Conclusions . DHEA-S achieves high serum levels after intraperitoneal injection without elevation of serum testosterone levels and produces no significant toxicity. Treatment with DHEA-S results in a significant reduction of proliferation of human pancreatic cancer cells in culture and when grown as subcutaneous tumors in athymic nude mice.