We have shown previously that norepinephrine induces migratory activity of tumor cells from breast, colon and prostate tissue via activation of beta-2 adrenergic receptors. Consequently, this effect can be inhibited pharmacologically by clinically established beta-blockers. Tumor cell migration is a prerequisite for metastasis formation, and accordingly we and others have shown that breast cancer patients, which take beta-blockers due to hypertension, have reduced metastasis formation and increased survival probability as compared to patients without hypertension or using other anti-hypertensive medication. Unlike the aforementioned tumor cells, pancreatic cancer cells show a reduced migratory activity upon norepinephrine treatment. By means of our three-dimensional, collagen-based cell migration assay, we have investigated the signal transduction pathways involved in this phenomenon. We have found that this conflicting effect of norepinephrine on pancreatic cancer cells is due to an imbalanced activation of the two pathways that usually mediate a pro-migratory effect of norepinephrine in other tumor cell types. Firstly, the inhibitory effect results from activation of a pathway which causes a strong increase of the secondary cell signaling molecule, cAMP. In addition, activation of phospholipase C gamma and the downstream protein kinase C alpha were shown to be already activated in pancreatic cancer cells and cannot be further activated by norepinephrine. We hypothesize that this constitutive activation of the phospholipase C gamma pathway is due to a cross-talk with receptor tyrosine kinase signaling, and this might also deliver an explanation for the unusual high spontaneous migratory activity of pancreatic cancer cells.
Excerpt on Cell Culture Protocol
The human breast cancer cell lineMDA-MB-468 and the pancreatic cancer cell lines CFPAC1,MiaPaCa2 and Panc1 were purchased from American Type Culture Collection (Manassas, VA, USA).The fourth pancreatic cancer cell line IMIM-PC2 was a gift from FranciscoX.Real (Spanish National Cancer Research Centre,Madrid,Spain). Pancreatic cancer cells were grown in Dulbecco's Modified Eagle Medium(DMEM,Sigma-Aldrich,Steinheim, Germany) supplemented with 10% foetal bovine serum (BiochromAG,Berlin,Germany), 2% penicillin/streptomycin (Sigma-Aldrich), 1m Msodiumpyruvate(Biochrom)and 2mM L-glutamine (PAALaboratories GmbH,Linz,Austria) at 37 1C in a humidified atmosphere of 5% CO2. MDA-MB-468 cells were cultivated in DMEM supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin at 37C in a humidified atmosphere of 5% CO2.
For the pancreatic cancer cell line where reference STR data are available(Panc-1,MIAPaCa-2,CFPAC-1),the GenomeLab Human STR PrimerSet(BeckmanCoulter,Krefeld,Germany)was used and analyzed on a CEQ8800 sequencer(BeckmanCoulter)according to the manufacturer's protocol to control for the cell line identity.STR data were submitted to on-line verification tool of DSMZ (German Collection of Microorganisms and Cell Lines) to confirm identity of human cell lines (http://old.dsmz.de/human_and_animal_cell_lines/main.php?contentleft_id=101). For IMIM-PC2,there is no reference available. Verification of the MDA-MB-468 was performed by the DSMZ.
The investigation of the migratory activity of the different cell lines was performed as described in detail previously . In short, from 4 to 8x 10^4 cells were mixed with carbonate-buffered collagen solution (1.63mg/ml collagen;NutaconBV,Leimuiden, the Netherlands) containing minimal essential medium (Sigma-Aldrich) and one or two of the following reagents: norepinephrine (10 mM; Sigma-Aldrich);atenolol(10 mM; Sigma-Aldrich); propranolol(10 mM; Sigma-Aldrich); the PKC activator phorbol 12-myristate13-acetate (PMA;50ng/ml;Sigma-Aldrich); the PKC inhibitor Gö6976(60nM;Calbiochem,BadSoden,Germany); the PLC activator 2,4,6 trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (3M3FBS;50 mM; Calbiochem); the PLC inhibitor U73122 (1 mM; Calbiochem);the myosin II inhibitor blebbistatin (100 mM; Sigma-Aldrich);the inhibitor of PTKs 4-amino-1-tert-butyl-3-(1′-naphthyl) pyrazolo [3,4–d] pyrimidine (PP1; 100nM;Calbiochem) and the inhibitor of PTKs4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine(PP2;50nM; Calbiochem); the PKA-inhibitor H89 (1 mM; New England Biolabs/Cell Signalling Technology,Frankfurt a.M.,Germany),and the cAMP-analogues 8Br-cAMP(1mM) and N6-Benzoyl-cAMP (6Bnz-cAMP; 100 mM; both provided by BIOLOG, Bremen, Germany),with the latter being a specific activator for the PKA. All of these substance were used at the same concentrations as in our previous studies with cell lines from other tumour entities,in order to make the results comparable.
After polymerization of the collagen,the migration of the cells was recorded by time-lapse videomicroscopy for 15h.Subsequently two-dimensional projections of the paths of 30 randomly selected cells were digitized in 15 min intervals. The migratory activity was calculated for each interval as the portion of cells, which was locomotory active within these 15min. The graphs show the time course of the migratory activity, the mean values and standard deviations. Statistically significant changes(po0.05) were calculated using Student's t-test(two-tailed and unpaired).