Human Pancreatic Adenocarcinoma PANC-1 Cells Research Articles

An L-fucose specific lectin from Aspergillus niger isolated from mycotic keratitis patient and its interaction with human pancreatic adenocarcinoma PANC-1 cells

An L-fucose specific lectin from pathogenic fungus Aspergillus niger isolated from the corneal smears of keratitis patient was purified in a single step using mucin coupled sepharose-4B column by 58-fold. The purified lectin, ANL has molecular mass of 30 kDa by SDS–PAGE and 31.6 kDa by ESI-MS. ANL is a glycoprotein with 2.59% carbohydrate. ANL is blood group nonspecific and also agglutinates rabbit erythrocytes. ANL is heat stable up to 50 °C and over a pH range of 7–10. Hapten inhibition studies revealed that ANL is specific to L-fucose, galactose, lactose and glycoproteins, showing highest MIC of 3.125 μg for L-fucose, mucin and fetuin. ANL has potent antibacterial activity against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and also it inhibits the biofilm formation by them. ANL showed strong binding to human pancreatic adenocarcinoma PANC-1 cells which was effectively blocked by L-fucose and mucin respectively by 76.2% and 84.2%. ANL showed dose and time dependent growth inhibitory effect on PANC-1 cells with IC50 of 1.25 μg/ml at 48 h. Effect of ANL was compared with another fucose specific lectin AOL, from Aspergillus oryzae showing an IC50 of 1.85 μg/ml at 48 h revealing promising clinical potential of ANL.

A Synthetic Analogue of Neopeltolide, 8,9-Dehydroneopeltolide, Is a Potent Anti-Austerity Agent against Starved Tumor Cells.

Neopeltolide, an antiproliferative marine macrolide, is known to specifically inhibit complex III of the mitochondrial electron transport chain (mETC). However, details of the biological mode-of-action(s) remain largely unknown. This work demonstrates potent cytotoxic activity of synthetic neopeltolide analogue, 8,9-dehydroneopeltolide (8,9-DNP), against starved human pancreatic adenocarcinoma PANC-1 cells and human non-small cell lung adenocarcinoma A549 cells. 8,9-DNP induced rapid dissipation of the mitochondrial membrane potential and depletion of intracellular ATP level in nutrient-deprived medium. Meanwhile, in spite of mTOR inhibition under starvation conditions, impairment of cytoprotective autophagy was observed as the lipidation of LC3-I to form LC3-II and the degradation of p62 were suppressed. Consequently, cells were severely deprived of energy sources and underwent necrotic cell death. The autophagic flux inhibited by 8,9-DNP could be restored by glucose, and this eventually rescued cells from necrotic death. Thus, 8,9-DNP is a potent anti-austerity agent that impairs mitochondrial ATP synthesis and cytoprotective autophagy in starved tumor cells.

PAR-3 Knockdown Enhances Adhesion Rate of PANC-1 Cells via Increased Expression of Integrinαv and E-Cadherin

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.

Proteinase-activated receptors differentially modulate in vitro invasion of human pancreatic adenocarcinoma PANC-1 cells in correlation with changes in the expression of CDC42 protein.

Proteinase-activated receptor-1 (PAR-1) and PAR-2 have been associated with increased invasiveness and metastasis in human malignancies. The role of PAR-3 has been less investigated. We examined the role of PARs in a human pancreatic adenocarcinoma PANC-1 cell line phenotype in vitro. We knocked down PAR-1, PAR-2, or PAR-3, whereas empty vector-infected cells served as controls. Specific peptide agonists of PARs were used to stimulate the receptors. In vitro assays of colony formation, migration, and invasion were used to characterize the phenotypes, and Western analysis was used to follow cell division control protein 42 homolog (CDC42) expression. PAR-1 and PAR-2 knockdowns (KDs) were markedly less, whereas PAR-3 KDs were robustly more migratory and invasive than the controls. Stimulation of PAR-1 or PAR-2 by their peptide agonists increased, whereas PAR-3 agonist reduced the invasion of the control cells. Knockdowns of all three PARs exhibited changes in the expression of CDC42, which correlated with the changes in their invasion. Conversely, stimulation of vector-control cells with PAR-1 or PAR-2 agonists enhanced, whereas PAR-3 agonist reduced the expression of CDC42. In the respective KD cells, the effects of the agonists were abrogated. The expression and/or activation of PARs is linked to the invasiveness of PANC-1 cells in vitro, probably via modulation of the expression of CDC42.

Serum Deprivation Induces Glucose Response and Intercellular Coupling in Human Pancreatic Adenocarcinoma PANC-1 Cells

This study aimed to investigate whether the previously described differentiating islet-like aggregates of human pancreatic adenocarcinoma cells (PANC-1) develop glucose response and exhibit intercellular communication. Fura 2-loaded PANC-1 cells in serum-free medium were assayed for changes in cytosolic free calcium ([Ca]i) induced by depolarization, tolbutamide inhibition of K(ATP) channels, or glucose. Dye transfer, assayed by confocal microscopy or by FACS, was used to detect intercellular communication. Changes in messenger RNA (mRNA) expression of genes of interest were assessed by quantitative real-time polymerase chain reaction. Proliferation was assayed by the MTT method. Serum-deprived PANC-1 cell aggregates developed [Ca]i response to KCl, tolbutamide, or glucose. These responses were accompanied by 5-fold increase in glucokinase mRNA level and, to a lesser extent, of mRNAs for K(ATP) and L-type calcium channels, as well as increase in mRNA levels of glucagon and somatostatin. Trypsin, a proteinase-activated receptor 2 agonist previously shown to enhance aggregation, modestly improved [Ca]i response to glucose. Glucose-induced coordinated [Ca]i oscillations and dye transfer demonstrated the emergence of intercellular communication. These findings suggest that PANC-1 cells, a pancreatic adenocarcinoma cell line, can be induced to express a differentiated phenotype in which cells exhibit response to glucose and form a functional syncytium similar to those observed in pancreatic islets.

Effects of New 4-Aryl-1,4-Dihydropyridines and 4-Arylpyridines on Drug Efflux Mediated by Multidrug Resistance-Associated Protein 1

The purpose of this study is to evaluate the effects of newly synthesized 4-aryl-1,4-dihydropyridine and pyridines on drug efflux mediated by multidrug resistance-associated protein 1 (MRP1, ABCC1). These compounds were designed to maximize inhibition of P-glycoprotein and minimize calcium channel binding activity, based on structure modifications of niguldipine. A [3H]vinblastine accumulation study was conducted in human small cell lung cancer H69AR (overexpressing MRP1) and wild type H69 cells. Five out of 16 dihydropyridines and 6 out of 9 pyridines were found to significantly increase the intracellular accumulation of vinblastine in resistant H69AR cells (p<0.01) at a concentration of 2.5 microM. Daunomycin accumulation studies, determined using a flow cytometric assay, were also performed in H69AR and human pancreatic adenocarcinoma Panc-1 cells and the results were highly correlated with those obtained from the [3H]vinblastine accumulation studies. Four compounds, which significantly increased vinblastine accumulation, were tested for their effect on daunomycin cytotoxicity in H69AR cells and found to significantly decrease the IC50 of daunomycin, confirming the accumulation study results. Compounds were also tested for their effect on intracellular glutathione (GSH) concentrations, a cosubstrate for MRP1-mediated efflux in H69AR and Panc-1 cells. No significant changes in the intracellular GSH level were observed in H69AR cells after treatment with these test compounds. However, following a 2-hr and 24-hr incubation with a dihydropyridine compound, Im, and its pyridine derivative IIm, there was a small (approximately 20%) but statistically significant decrease in intracellular GSH in Panc-1 cells. Our results indicate that some dihydropyridine and pyridine compounds in our series could inhibit MRP1-mediated transport and that GSH modulation plays a minor, if any, role in this effect.