Abstract
Simple SummaryAnandamide (AEA) belongs to the group of endocannabinoids and possesses various regulatory properties in physiological as well as pathological processes occurring in the organism. In this research some basic biological tests were applied to investigate AEA-induced changes in cell metabolism and motility, as well as advanced biophysical methods for the determination of the differences in the cell glycosylation profile on a highly dangerous model of melanoma skin cancer, for which an effective therapy is not yet available. Our research suggests that anandamide treatment of metastatic melanoma cells increases the cell metabolism which leads to the reduction in the metastatic potential of cells in terms of the cell glycosylation profile and cell migration. In the view of our research, it can be presumed that anandamide usage in the combined therapy of advanced melanoma would be an advantage for the patient.An effective therapy for advanced melanoma, a skin cancer with the highest mortality, has not yet been developed. The endocannabinoid system is considered to be an attractive target for cancer treatment. The use of endocannabinoids, such as anandamide (AEA), is considered to be much greater than as a palliative agent. Thus, we checked its influence on various signaling pathways in melanoma cells. Our investigation was performed on four commercial cell lines derived from different progression stages (radial WM35 and vertical WM115 growth phases, lymph node WM266-4 metastasis, solid tumor A375-P metastasis). Cell viability, glucose uptake, quantification of reactive oxygen species production, expression of selected genes encoding glycosyltransferases, quantification of glycoproteins production and changes in the glycosylation profile and migration, as well as in cell elastic properties were analyzed. The cell glycosylation profile was investigated using the biophysical profiling method—the quartz crystal microbalance with dissipation monitoring (QCM-D). Anandamide treatment of only metastatic cells resulted in: an increase in the cell metabolism, a decrease in GFAT-1 and DPM1 expression, followed by a decrease in L1-CAM glycoprotein production, which further influenced the reduction in the cell glycosylation profile and migration. Considering our results, AEA usage is highly recommended in the combined therapy of advanced melanoma.
Highlights
Most of the diagnosed melanoma skin cancers are developed from melanocytes due to the ultraviolet (UV) radiation-related induction of genetic mutations, immunosuppression and photoaging [1]
After 24 h incubation of melanoma cells with 0.05–10 μM AEA, the number of melanoma cells stained with crystal violet was comparable to the control in the whole range of concentrations (~90% cells even for the highest AEA concentration; Figure 1A), but the mitochondrial dehydrogenase activity of melanoma cells was decreased by approximately 25% for 10 μM AEA (Figure 1B)
The double FDA/PI staining of closely related melanoma cell lines revealed that metastatic WM266-4 cells are more sensitive to the narrowed range 0.5–4 μM of AEA concentration than WM115 cells from the primary site (Figure 1C)
Summary
Most of the diagnosed melanoma skin cancers are developed from melanocytes due to the ultraviolet (UV) radiation-related induction of genetic mutations, immunosuppression and photoaging [1]. The vertical growth phase (VGP) is considered the most crucial moment in the process of melanoma development. These cells gain the ability to survive in the dermis and to proliferate there. If these cells acquire a further ability to metastasize, the patient has minimal chances for long-term survival (more than a few months). This is because melanoma cells can spread through the lymph nodes and vessels; it is difficult for the tumor cells to be eliminated from the whole organism [6–8]. That is why treatment allowing the slowdown of the migration or blocking the cells in the primary growth phases could help the patient before surgery can be performed
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