Repurposing riluzole as an anti-osteosarcoma agent

Abstract

We have studied riluzole, a glutamate-release inhibitor, as a novel anti-osteosarcoma agent. YAP (Yes-associated protein) is recruited by Bax promoter to stimulate its expression during riluzole-induced apoptosis in the human metastatic osteosarcoma cell line LM7. Given the substantial genetic heterogeneity in osteosarcoma, studies on the efficacy of riluzole in diverse osteosarcomas will be an asset in developing preclinical studies. Toward this goal, we investigated the effects of riluzole on 11 osteosarcoma cell lines derived from primary or metastatic tumors of mouse or human origin and on four independent patient-derived xenograft (PDX) tumor cell lines. We found that most of the osteosarcoma cell lines, including PDX cell lines secrete glutamate and exhibit invasive abilities. Cell growth and invasive ability of all the cell lines and PDX cell lines are inhibited by riluzole. Additionally, riluzole suppresses the activity of matrix metalloprotease-2 (MMP2) in most of the osteosarcoma cell lines (but not the PDX cells). These results suggest that riluzole's inhibitory effects on osteosarcoma invasion may in part be attributable to the inhibition of MMP2 activity, and that riluzole is potentially an effective agent for inhibiting growth of primary and metastatic osteosarcomas with a wide range of genetic profiles.

Keywords: MMP2; metastasis; osteosarcoma; patient-derived xenograft cell lines; reactive oxygen species; riluzole.

Figure 1

p53 expression and glutamate secretion in OS cells. (A) Western blots showing expression of p53 in OS cell lines and PDX cells. The data was analyzed by one-way ANOVA followed by Dunnett’s test. *p<0.05. (B–D) Glutamate secreted into media between days 0 and 7 by primary OS cell lines, hFOB and metastatic cell lines, and hFOB and PDX cells. The experiment was repeated three times; the average of the three is shown. The data was analyzed by one-way ANOVA followed by Dunnett’s test for each day, with the value for hFOB for that day serving as the comparison value. *p <0.05, **p<0.01, ***p<0.001. (E–F) mRNA expression of mGluR1 and mGluR5 by OS cell lines, measured by quantitative PCR. *p<0.05, **p<0.01, ***p<0.001. (G–H) Expression of vGLUT and xCT by OS cell lines and PDX cells, measured by western blot. The data was analyzed by one-way ANOVA followed by Dunnett’s post hoc analysis. ***p<0.001.

Figure 2

Inhibition of OS cell growth. Percentage inhibition was measured using an MTT assay in all of the OS cell lines using riluzole concentrations of 0, 5, 10, 25, 50, and 100 µM. Cell lines shown are (A) hFOB, (B) OS482, (C) K7M2, (D) MG63, (E). MG63.3, (F) SaOS2, (G) LM7, (H) HOS, (I) HOS/MNNG, (J) 143B, (K) U2OS, (L) OS24, (M) OS29, (N) OS33, and (O) OS69. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc analysis. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Riluzole treatment increases intracellular ROS except 143B and OS69. ROS production in the cell lines were measured using the DCFH-DA method. The cells were treated with either DMSO (negative control), riluzole 50 µM, riluzole 100 µM, H₂O₂ 50 µM, or H₂O₂ 100 µM (positive control). Cell lines shown are (A) hFOB, (B) SaOS2, (C) LM7, (D) U2OS, (E) MG63, (F) MG63.3, (G) HOS, (H) 143B, (I) OS24, (J) OS29, (K) OS33, and (L) OS69. The experiments were repeated in triplicates with three different biological samples. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc analysis. *p<0.05, **p<0.01, ***p<0.001.

Figure 4

The suppression of invasive abilities in OS cell lines by riluzole. Boyden chamber invasion assay was performed to assess the invasive abilities of OS cell lines. The invasive abilities of (A) MG63, (B) MG63.3, (C) SaOS2, (D) LM7, (E) U2OS, (F) HOS, (G) HOS-MNNG, (H) 143B, (I) OS24, (J) OS29, (K) OS33, and (L) OS69 were examined in the presence of culture media containing 1% FBS. The cells were treated either with DMSO (control) or with riluzole at 25 µM. Media with 0.1% FBS was used as a negative control (−). Magnification bar in the image represents 100 μm. Statistical significance was determined using Student’s t-test. *p<0.05, **p< 0.01, ***p<0.001.

Figure 5

The inhibition of MMP2 activity in OS cell lines by riluzole. Gelatin zymography analysis was used to examine the activity of MMP2 and MMP9 secreted by (A) LM7, (B) MG63, (C) MG63.3, (D) HOS, (E) HOS-MNNG, (F) 143B, (G) OS24, (H) OS29, (I) OS33, and (J) OS69 cells. The cells were treated with either DMSO (control) or riluzole at 25 µM. The band intensity was quantified using ImageJ. All experiments were independently repeated three times. Statistical significance was determined using Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.

Figure 6

MMP2 activity is transcriptionally regulated by riluzole. Quantitative PCR was performed to measure mRNA levels for MMP2 and MMP9 using total RNA from cells grown in 0.1% FBS and either not treated or treated with 25 μM riluzole for 48 hours. Cell or PDX lines shown are (A) LM7, (B) MG63, (C) MG63.3, (D) HOS, (E) HOS-MNNG, (F) 143B, (G) OS24, (H) OS29, (I) OS33, and (J) OS69. Statistical significance was determined using Student’s t-test. *p<0.05, **p<0.01.