PMC

Riluzole combined with rapamycin effectively decreases tumor burden in xenograft model. C8161^{BRAF/NRAS/PTEN WT} and UACC903^{BRAFV600E,PTENMUT} cells were subcutaneously injected into both flanks of 20-g, 6-week-old male, NC NU/NU mice. Mice underwent treatment with either vehicle (DMSO), riluzole (7.5 mg/kg), rapamycin (1 mg/kg), or riluzole (7.5 mg/kg) combined with rapamycin (1 mg/kg). The final tumor volumes in each treatment arm are shown in a box plot for C8161 (A) and UACC903 (B). The combination of riluzole and rapamycin was statistically different than single therapy by ANOVA (P < .05) in each xenograft.

Steady-state effects of riluzole combinatorial therapy on the PI3K/MAPK pathways. C8161, UACC903, HT144, SKMEL2, and UACC930 human melanoma cell lines were plated at 8 × 10⁵ on a 60-mm dish and allowed to recover o/n. The following day, cells were treated with either vehicle (DMSO), 10 μM riluzole, 1 μM API-2, 2.5 nM rapamycin, 10 μM riluzole combined with 1 μM API-2, or 10 μM riluzole combined with 2.5 nM rapamycin for 18 hours. The 18-hour treatment time point was chosen because previous work has shown that that is adequate time for feedback loop activation to take place in melanoma cell lines in vitro and is representative of steady state (A). Band intensities were analyzed with Gene Tools software (Syngene). Raw volume of pAkt and total Akt with background subtracted was normalized to GAPDH, with vehicle-treated cells set as 100%. The mean ratio of pAkt/Total Akt for C8161 (triplicate) and UACC903 (duplicate) is shown for each single-agent and combinational therapy (B).

Tumor PI3K pathway signaling with combinational riluzole therapy compared with nanoculture (3D) modeling. Effects on the PI3K pathway in each xenograft model (C8161 or UACC903) were determined with each treatment: vehicle, riluzole, rapamycin, or combinational therapy. A representative sample of each treatment arm is displayed from both tumor cell lines. pAKT, total AKT, and GAPDH expression levels were quantified using Gene Tools software (Syngene) for each xenograft tumor shown in A. For each cell line, the pAKT/total AKT is reported, normalized to GAPDH with vehicle (DMSO) treated reported as 100% (B). UACC903 cells were also plated into the nanoculture plate system and allowed to form spheroids for 2 days. Afterward, cells were treated with (I) vehicle, (II) 10 μM riluzole, (III) 2.5 nM rapamycin, and (IV) combinational treatment for 18 hours (C). Lysate from the nanoculture plate experiments was collected and underwent Western blot analysis to look at changes in the PI3K pathway (D). Results revealed that nanoculture (3D) modeling of combinational drug treatment significantly resembled in vivo xenograft results.

Riluzole combined with API-2 and rapamycin inhibits anchorage-independent growth of melanoma cell lines. Soft agar colony assays were performed with C8161 (1 × 10⁴ cells), UACC903 (1.5 × 10⁴ cells), HT144 (1.5 × 10⁴ cells), SKMEL2 (1.5 × 10⁴ cells), and UACC930 (5 × 10⁴ cells) in the presence of one of the following low doses of inhibitors: vehicle (DMSO), 10 μM riluzole, 1 μM API-2, 2.5 nM rapamycin, 10 μM riluzole combined with 1 μM API-2, or 10 μM riluzole combined with 2.5 nM rapamycin. Three representative photographs were taken of each treatment (A). The number of colonies from three representative photomicrographs was totaled and colony-forming potential relative to DMSO-treated cells was determined (B) as well as the size distribution of colonies (C). Experiments were repeated with each cell line three times, with the exception of UACC930 (negative control, one experiment). ANOVA revealed that there is a significant difference between the colony treatment groups across cell lines (F(5,84) = 75.591, P < .001). Scheffé post hoc test indicates that each combinatorial treatment had a statistically significant difference compared to single treatment or DMSO across cell lines (P < .05).