Anandamide-Modulated Changes in Metabolism, Glycosylation Profile and Migration of Metastatic Melanoma Cells

Abstract

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.

Keywords: anandamide; biophysical methods; glycosylation profile; lectin–glycan interaction; melanoma metastasis; metabolism; migration.

Figure 1

Influence of AEA on the number of melanoma cells calculated from CV assay (A), the mitochondrial dehydrogenase activity of melanoma cells checked by means of the MTT assay (B) and living/dead melanoma cell estimation based on the double FDA/PI staining for the primary VGP site WM115 cells—red (C) and metastatic WM266-4 cells—blue (D).

Figure 2

Influence of AEA on melanoma cell metabolism: measurements of the glucose uptake (A) and quantification of ROS production (B). The investigated cell lines are from the following stages of melanoma progression: RGP site (WM35—yellow), VGP site (WM115—red), metastasis to the lymph node (WM266-4—blue) and from the solid tumor site (A375-P—violet). Two additional controls were prepared: a positive control—cells treated with 100 μM tBHP and a negative control—cells treated with 5 mM GSH. The statistical significance of the p-value below 0.05 (*) was marked on the graph.

Figure 3

RT-qPCR analysis of GFAT-1 (A) and DPM1 (B) expression in AEA-treated melanoma cells. The investigated cell lines are from the following stages of melanoma progression: RGP site (WM35—yellow) and from the solid tumor site (A375-P—violet). The statistical significance of the p-value below 0.05 (*) was marked on the graph.

Figure 4

Difference in the L1-CAM glycoprotein production by AEA-treated melanoma cells. The investigated cell lines are from the following stages of melanoma progression: RGP site (WM35) and from the solid tumor site (A375-P). The statistical significance of the p-value below 0.05 (*) was marked on the graph.

Figure 5

QCM-D results of the lectin–glycan binding measurements performed on AEA-treated and closely related (from the same patient) VGP cells (A) WM115 in red) and cells from the metastasis to the lymph node (B) WM266-4 in blue). The corresponding relations of the reverse in relaxation time plotted as a function of lectin Con A concentration with the established linear regressions were used to deliver the kinetic parameters of the lectin-glycan interaction (k_on and k_off).

Figure 6

The results of the lectin-ELISA assay for AEA-treated and closely related (from the same patient) melanoma VGP cells ((A) WM115 in red) and cells from the metastasis to the lymph node ((B) WM266-4 in blue), where the fluorescent intensity was plotted as a function of the bound lectin Con A-FITC to the glycans present on the investigated cells.

Figure 7

Df plots received for the 6.4 μM Con A concentration of the lectin-glycan interaction performed on AEA-treated melanoma RGP cells (A)—WM35), VGP cells (B)—WM115), metastasis to the lymph node (C)—WM266-4) and solid tumor metastatic cells (D)—A375-P). (E)—Overview of the obtained viscoelastic index values for Con A concentrations (1.6–12.8 μM) interacting with glycans of melanoma cells treated with AEA. The statistical significance of the p-value below 0.05 (*) was marked on the graph.

Figure 8

Migration rate analysis of melanoma cells: (A)—the representative pictures of the closely related WM115 and WM266-4 melanoma cells (from the same patient) after creating the scratch (0 h) and 24 h later after the 1 μM AEA treatment (scale bar—100 μm). (B)—the calculated migration rate of different melanoma cells after 1 μM and 5 μM AEA treatment. The statistical significance of the p-value below 0.05 (*) was marked on the graph.

Figure 9

(A)—the calculated elastic modulus of different melanoma cell lines treated with 1 μM AEA at the indentation depth of 300 nm. Cell lines: WM35—primary RGP site; WM115—primary VGP site; WM266-4—metastasis to the lymph node; A375-P—solid tumor metastatic site. The representative pictures of A375-P melanoma cells: control (B) and treated with 1 μM AEA (C) after cell staining with phalloidin labeled with Alexa Fluor 488 (ex. 495 nm, em. 518 nm) and Hoechst 33342 (ex. 350 nm, em. 461 nm).