Cytoplasmic Ca2+ concentration changes evoked by muscarinic cholinergic stimulation in primary and metastatic melanoma cell lines

Abstract

Experiments were performed to explore differences between cultured primary and metastatic melanoma cell lines in their muscarinic acetylcholine receptor-mediated intracellular Ca signalization. The expression of type 1 and type 3 muscarinic receptors was detected and compared at the protein level using both immunocytochemistry and semiquantitative western blotting. The functionality of muscarinic receptors was tested by applying carbamylcholine (CCh; 1 mmol/l) and by recording the associated increases in cytoplasmic Ca using Ca imaging with the application of the Ca indicator dye, fluo-4. These data indicate that the expression levels of the receptor proteins were not significantly different in the metastatic (HT199, HT168-M1) and the primary (WM35) cell lines. Although Ca transients were evoked in all the three cell lines by CCh, the proportion of the CCh-positive cells was smaller amongst the WM35 cells. The Ca transients could be effectively blocked by atropine (0.1 mmol/l). The time courses of the Ca transients were highly variable, and in some instances they showed a late (plateau-like) component whose presence crucially depended on the influx of extracellular Ca. When the extracellular Ca concentration was reduced, the duration of the CCh-evoked transients was considerably decreased; a phenomenon that was more pronounced in the metastatic cell lines. Although there are no fundamental differences in the muscarinic receptor-mediated Ca signalization of the primary and metastatic cell lines, the quantitative differences showed in this study may partially explain the increased malignancy and migratory potential of the metastatic cells.

Exploring Differential Connexin Expression across Melanocytic Tumor Progression Involving the Tumor Microenvironment

“…Human invasive melanoma cell lines A2058 (metastasis from brain) [50], HT199 (metastatic to the liver and lung) [51], and WM983/A (vertical growth phase) [52,53], were available in the tumor bank of the 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest. They were grown in RPMI 1640 medium (PAN-Biotech GmbH, Aidenbach, Germany) supplemented with 5% fetal bovine serum (FBS) and 1% gentamycin.…”

Breast cancer: Muscarinic receptors as new targets for tumor therapy

“…Although all of them responded to carbamylcholine, increasing cytosolic calcium levels, the metastatic cell line responded with a higher peak in calcium concentration. The authors speculated that this difference could be responsible for a higher malignance and migratory potential in metastasis[ 142 ]. In accordance with the latter observations, we have reported that the expression of mAChRs is significantly higher in the metastatic murine mammary cell line LMM3 than in the non-metastatic LM3 tumor cells that originated them[ 82 ].…”

Invasiveness‐triggered state transition in malignant melanoma cells

“…Subsequently, monoclonal antibody M35 staining results confirmed primarily the expression of muscarinic receptors in melanoma cells (Boss, Oppitz, Lippert, & Drews, ; Lammerding‐Köppel et al, ; Noda, Lammerding‐Köppel, Oettling, & Drews, ; Oppitz, Busch, Garbe, & Drews, ). Nagy et al () showed M3 in different melanoma cell lines by both immunocytochemistry and western blot methods (Nagy et al, ). Interestingly, Lammerding‐Koppel et al () found different intensities of the muscarinic receptor in melanoma tissue.…”