The Angiotensin II Type 1 Receptor Antagonist Losartan Affects NHE1-Dependent Melanoma Cell Behavior

Daniel Navin Olschewski, Verena Hofschröer, Nikolaj Nielsen, Daniela G. Seidler, Albrecht Schwab, Christian Stock
2018 Cellular Physiology and Biochemistry  
Background/Aims: The peptide hormone angiotensin II (ATII) plays a prominent role in regulating vasoconstriction and blood pressure. Its primary target is the angiotensin II receptor type 1 (AT 1 ), the stimulation of which induces an increase in cytosolic [Ca 2+ ] and calmodulin activation. Ca 2+ -bound (activated) calmodulin stimulates the activity of the Na + / H + exchanger isoform 1 (NHE1); and increased NHE1 activity is known to promote melanoma cell motility. The competitive AT 1
more » ... titive AT 1 receptor inhibitor losartan is often used to lower blood pressure in hypertensive patients. Since AT 1 mediates ATII-stimulated NHE1 activity, we set out to investigate whether ATII and losartan have an impact on NHE1-dependent behavior of human melanoma (MV3) cells. Methods: ATII receptor expression was verified by PCR, F-actin was visualized using fluorescently labeled phalloidin, and cytosolic [Ca 2+ ] and pH were determined ratiometrically using Fura-2 and BCECF, respectively. MV3 cell behavior was analyzed using migration, adhesion, invasion and proliferation assays. Results: MV3 cells express both AT 1 and the angiotensin II receptor type 2 (AT 2 ). Stimulation of MV3 cells with ATII increased NHE1 activity which could be counteracted by both losartan and the Ca 2+ / calmodulin inhibitor ophiobolin-A. ATII stimulation induced a decrease in MV3 cell migration and a more spherical cell morphology accompanied by an increase in the density of F-actin. Independently of the presence of ATII, both NHE1 and migratory activity were reduced when AT 1 was blocked by losartan. On the other hand, losartan clearly increased cell adhesion to, and the invasion of, a collagen type I substrate. The AT 2 inhibitor PD123319 did not affect NHE1 activity, proliferation and migration, but increased adhesion and invasion. Conclusion: Losartan inhibits NHE1 activity and the migration of human melanoma cells. At the same time, losartan promotes MV3 cell adhesion and invasion. The therapeutic use of AT 1 antagonists (sartans) in hypertensive cancer patients should therefore be given critical consideration. Olschewski et al.: Losartan Effects on Melanoma Cells consists of chemotherapeutics combined with antibodies. Thus, the question arises as to how AT 1 inhibitors such as losartan contribute to melanoma treatment. Materials and Methods Cells and cell culture For all experiments we used human melanoma cells of the MV3 line [20] . Cells were kept in a humidified atmosphere with 5% CO 2 / 95% air at 37 °C (Hera-Cell 150, Heraeus, Germany) and used for experiments at passages 3-10. RPMI-1640 (Sigma-Aldrich, Taufkirchen, Germany) with L-glutamine, NaHCO 3 and 10% FCS (fetal calf serum, PAA Laboratories GmbH, Pasching, Germany) served as culture medium. Reverse transcription-PCR (RT-PCR) RT-PCR was applied to verify the presence of AT 1 and AT 2 in MV3 cells. Total RNA was isolated from MV3 cells using the TRIzol Reagent (Invitrogen, Waltham, USA) according to the manufacturer's instructions. 1µg of RNA was transcribed into cDNA using the SuperScript® III Reverse Transcriptase kit (RT; Invitrogen) according to the protocol of the manufacturer. The primer pairs were designed accordingly to the sequences used by Malendowicz et al. 2000 [21] (Metabion). Sequences were for AT 1 : forward, 5'-GATGATTGTCCCAAAGCTGG-3'; reverse, 5'-TAGGTAATTGCCAAAGGGCC-3' and for AT 2 : forward, 5'-TTCCCTTCCATGTTCTGACC-3'; reverse, 5'-AGAAGCTCCGCAGTGTGTTT-3'. GAPDH was used as a normalization control. For both AT 1 and AT 2 , every PCR was launched with a denaturing incubation for 10 min at 95°C. This step was followed by 30 cycles as described: [30 sec at 94°C, 60 sec at 60°C and 60 sec at 72°C]×30, followed by 10 min at 72°C. 1.5% agarose (Serva, Heidelberg, Germany) gel electrophoresis and Sybr-Gold (SYBRÒ Gold Nucleic Acid Gel Stain, Invitrogen) were used to analyze the PCR results. Exposure to UV-light and visualization of the bands were conducted in a documentation chamber (Bio-Rad Laboratories GmbH, Munich, Germany). In order to exclude possible DNA contaminations "no-amplification-controls" (=RTcontrols) were performed employing the same protocol and the same reagents except the reverse transcriptase. Quantification of F-actin by phalloidin MV3 cells were seeded on collagen I coated cover slips (Collagen G solution (Biochrom GmbH, Berlin, Germany) at a dilution of 1:10 (v/v) in PBS (PBS contained in mmol l -1 : 137 NaCl, 2.1 KCl, 8.1 Na 2 HPO 4 and 1.76 KH 2 PO 4 ) and were allowed to attach and spread for 2 h in FCS-containing RPMI-medium in a humidified atmosphere. The cells were then incubated overnight with FCS-free medium. The next day, they were exposed to ATII (100 nmol l -1 ) and/or losartan (0.7 µmol l -1 ) for 2 h in a humidified atmosphere and then fixed with 3.5% (w/v) paraformaldehyde in PBS. The cell membranes were permeabilized by a 20 min exposure to 0.1% (v/v) Triton in PBS. Non-specific binding sites were blocked using 3% (w/v) Bovine Serum Albumin (BSA) in PBS for 2 h at room temperature. A dilution of 1:250 of Alexa Fluor® 488-phalloidin (Invitrogen) was used for selectively staining F-actin in MV3 cells. After a 1 h incubation with the phalloidinconjugate, the cover slips were washed in PBS, covered with Dako fluorescence mounting medium (Dako, Carpinteria, USA) and then put upside down on glass slides. The actin staining was evaluated using an inverted microscope (Axiovert200, Carl Zeiss, Inc., Göttingen, Germany), a digital camera (Model 9.0, RT-SE-Spot, Visitron Systems) and the MetaVue software. The NIH ImageJ software (http://rsb.info.nih.gov/ ij/) was used to determine the area and the mean fluorescence intensity of the labeled cells. To correct height-and morphology-dependent effects on fluorescence intensity, the area of a cell was multiplied by its mean fluorescence intensity. All images were taken at the same exposure time (750 ms). Three independent trials were performed and ten images (=cells) per trial and condition were evaluated. Values are given in arbitrary units. Measurement of intracellular Calcium [Ca 2+ ] i Ratiometric measurements of the intracellular Ca 2+ concentration [Ca 2+ ] i were performed employing the Ca 2+ indicator Fura-2 (Invitrogen). MV3 cells were plated onto collagen coated coverslips and allowed to adapt for at least 3 h. Prior to each measurement, cells were incubated for one hour in HEPES-buffered Ringer's solution containing (mmol l -
doi:10.1159/000488274 pmid:29558744 fatcat:u735wihxnzfutozozylsr6stp4