The glutamate release inhibitor Riluzole decreases migration, invasion, and proliferation of melanoma cells

Abstract

The goal of this study was to examine the effects of metabotropic glutamate receptor-1 (GRM1) blockade on melanoma anchorage-independent growth and invasion. We performed colony and invasion assays using GRM1-expressing melanoma lines and the GRM1-negative UACC930 line. Using the glutamate-release inhibitor Riluzole or the non-competitive GRM1 antagonist BAY 36-7620 we were able to induce considerable inhibition of colony formation and invasion in GRM1-expressing melanoma lines. Neither pharmacological agent induced significant reduction in colony formation or invasion in the GRM1-negative melanoma line, UACC930. Additionally we assessed the efficacy of these inhibitors to inhibit the growth of fresh melanoma tumor samples cultured on a 74-mum nylon mesh. Both Riluzole and BAY 36-7620 significantly inhibited tumor cell growth into the interstitial spaces of the mesh. When repeated with normal mole samples both inhibitors were much less effective in preventing the outgrowth of cells. These experiments show that a specific antagonist of GRM1 (BAY 36-7620) or an inhibitor of glutamate release (Riluzole) can significantly suppress melanoma migration, invasion and colony formation as well as inhibit the proliferation of fresh melanoma cells. These findings, added to our previous work, strengthen the case that GRM1 is a valid therapeutic target in patients with melanoma.

Figure 1. Riluzole and Bay 36-7620 inhibit the anchorage independent growth of human melanoma cell lines

Soft agar colony assays were performed with C8161 (5 × 10⁴ cells), HT144 (1.5 × 10⁴ cells), SKMEL2 (1.5 × 10⁴ cells) and UACC930 (5 × 10⁴ cells) in the presence of 25μM Riluzole, 50μM Bay 36-7620, 10μM U0126 or vehicle solvent (DMSO). Colony assays were fed with 200μL of 10% FBS RPMI containing the appropriate inhibitors every 48 hrs after the initiation of the assay. Photomicrographs of 3 representative fields were taken after 14 days or 28 days in the case of UACC930 (a.). The number of colonies (b.) and colony size (c.) for each photomicrograph was determined using Image J and the totals plotted. Histograms represent the average of 3 independent experiments with the exception of UACC930 (2 experiments). Manification bar = 500μm (a.)

Figure 2. Glutamate blockade inhibits the invasive potential of human melanoma cell lines

Boyden chamber invasion assays were performed with various human melanoma cell lines as described in the materials and methods in the presence of 25μM Riluzole, 50μM Bay 36-7620, 10μM U0126 or vehicle solvent (DMSO). 1 × 10⁵ cells were used for the assays with the exception of C8161 and A2058 cell lines (2 × 10⁴ cells). Cells were allowed to invade 18 hrs with the exception of UACC930 (48 hrs). For UACC930 a fluid renewal containing inhibitors was performed at 24hrs. Invaded cells were fixed, stained and 3 representative fields were photomicrograhed (A.). The relative levels of invasion for each cell line and treatment was determined with Image J and the results plotted (B.). The histogram represents the average of 3 independent experiments. For uPA activity assays (C.), 5 × 10⁵ cells were plated into 4 parallel wells in a 6 well plate and allowed to recover O/N. They were then treated with the indicated inhibitors in serum free RPMI 16–40 media for 6 hrs, then replaced with fresh serum free media containing the inhibitors and returned to the incubator for an additional 12 hrs. Media was then collected and protein lysates prepared as indicated in the materials and methods. Using 20μg of total cell lysate, an invitro uPA activity assay was performed according to the manufacturer (C.). Manification bar = 500μm (A.)

Figure 3. Treatment of human melanoma cells with Riluzole inhibits AKT and ERK phosphorylation

C8161, HT144, SKMEL2 and UACC930 human melanoma cell lines were plated at a density of 8 × 10⁵ cell/60mm dish and allowed to recover O/N. The next day the cells were treated 25μM Riluzole or vehicle solvent (DMSO) for 8hrs after which total cell lysates (TCL) were prepared, protein levels determined and 10μg of TCL was resolve by SDS-PAGE (A.). Proteins were electroblotted to PVDF membranes and probed with the indicated primary and corresponding secondary antibodies. Chemiluminescent detection was performed via ECL-plus or ECL-Advance and exposure to X-ray film. The representative of 2 (UACC930) or 3 independent experiments is shown (A.). Densitometric analysis was performed using Image J to determine the protein phosphorylation and expression levels. The normalized protein phosphorylation levels were then plotted (B.) The histogram represents the average of 2 (UACC930) or 3 independent experiments. A parallel set of cells were treated as above, however total cell lysates (TCL) were prepared at 18 hrs post-treatment and processed as above. (C.).

Figure 4. Riluzole and Bay 36-7620 inhibit the growth of fresh melanoma tumors

Freshly harvested melanoma tissues were used in an organotypic growth assay as indicated in the materials and methods. 24 hrs after implanting the tumor tissue on the mesh the samples were treated with 25μM Riluzole, 50μM Bay 36-7620, 10μM U0126 or vehicle solvent (DMSO). Media was replenished every 48 hrs. Cells were allowed to grow out from the tumor sample for a period of 14 days after which photomicrographs were taken. Original tumor sample is on the left of each photomicrograph.