A disintegrin-like and metalloproteinase 15 facilitates glioblastoma proliferation and metastasis through activation of the protease-activated receptor 1

Abstract

Objective: Glioblastoma hinders therapeutic interventions and prognostic outlooks. At the same time, a disintegrin-like and metalloproteinase 15 (ADAM15) influences cellular processes, such as adhesion and migration. Furthermore, protease-activated receptor 1 (PAR1), a vital receptor, impacts tumorigenesis and disease progression. This study aimed to investigate ADAM15 and PAR1 interaction in epithelial-mesenchymal transition (EMT) modulation in glioblastoma behavior and provide insights into therapeutic targets.

Material and methods: The impacts of ADAM15 overexpression and PAR-1/2 inhibition on the proliferation, invasion, and migration of glioblastoma cells U251 and U87 were evaluated using transwell assays, EdU incorporation, clonogenic assay, Ki67 immunohistochemistry, and immunofluorescence staining. Real-time quantitative polymerase chain reaction and Western blot analysis were employed to investigate the impact of ADAM15 on PAR1 expression.

Results: After analyzing the impacts of ADAM15 overexpression on the migration, invasion, and proliferation of human glioblastoma cell lines U251 and U87, the results showed that ADAM15 overexpression significantly enhanced migration (P < 0.001) and invasion rates (P < 0.001), as confirmed by scratch and transwell assays, thus indicating its tumor-promoting effects. This study revealed a significant increase in colony formation (P < 0.001), EdU incorporation (P < 0.001), and Ki67-positive cells (P < 0.001) in the ADAM15 overexpressed group. PAR1 and EMT markers were significantly increased in the ADAM15 overexpressed group (P < 0.001). Treatment with the PAR-1 antagonist SCH79797 inhibited EMT (P < 0.01) and suppressed cell proliferation (P < 0.001), migration (P < 0.001), and invasion (P < 0.001) in U251 and U87 cells overexpressing ADAM15, indicating the involvement of PAR-1 signaling in the effects of ADAM15 on cell behaviors. In comparison, the PAR-2 antagonist FSLLRY-NH2 did not show significant effects on EMT or these cell behaviors.

Conclusion: ADAM15 drives glioblastoma cell lines U251 and U87 progression through PAR1.

Keywords: A disintegrin-like and metalloproteinase domain 15; Epithelial-mesenchymal transition; Protease-activated receptor 1; glioblastoma.

Figure 1:

Impacts of ADAM15 overexpression on tumor migration, invasion, and proliferation. (a and b) Measurement of ADAM15 protein expression. (c) Measurement of ADAM15 mRNA expression. (d) Evaluation by scratch assay, Scale bar = 100 μm. Objective: 100×. (e) Relative migration rate in U251 and U87 for scratch assay. (f) Evaluation of U251 and U87 migration for 12 h through transwell assay, Scale bar = 50 μm. Objective: 200×. (g) Migrated cells per field in U251 and U87 for the transwell assay. (h) Evaluation of transwell assay, with Matrigel in U251 and U87 invasion for 72 h. Scale bar = 50 μm. Objective: 200×. (i) Relative invasion rate in U251 and U87 for the transwell assay. n = 5 independent replicates. ^✶✶✶P < 0.001. ADAM15: A disintegrin-like and metalloproteinase 15, mRNA: Messenger RNA.

Figure 2:

Impact of ADAM15 overexpression on the proliferation ability of glioblastoma cells. (a) Measurement of colony number in U251 and U87. (b) Clonogenic assay in U251 and U87. (c) EdU incorporation in U251 and U87. Scale bar = 20 μm. Objective: 400×. (d) Proliferation rates in U251 and U87 for EdU incorporation. (e) Evaluation of Ki67 immunohistochemistry staining. Scale bar = 20 μm. Objective: 400×. (f) Ki67 positive cell rates in U251 and U87 for Ki67 immunohistochemistry staining. n = 5 independent replicates. ^✶✶✶ P < 0.001. ADAM15: A disintegrin-like and metalloproteinase 15.

Figure 3:

Impacts of ADAM15 overexpression on PAR1 expression and EMT. (a and b) Measurement of PAR1 protein expression in U251 and U87. (c) Measurement of PAR1 mRNA expression in U251 and U87. (d) Measurement of E-cadherin, N-cadherin, and Vimentin protein expression in U251 and U87. (e) Relative E-cadherin protein expression in U251 and U87. (f) Relative N-cadherin protein expression in U251 and U87. (g) Relative Vimentin protein expression in U251 and U87. (h) Immunofluorescence of E-cadherin and Vimentin staining in U251 and U87. Scale bar = 20 μm. Objective: 400×. n = 5 independent replicates. ^✶✶✶ P < 0.001. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1, Epithelial-mesenchymal transition, mRNA: Messenger RNA.

Figure 4:

Inhibition of ADAM15-induced proliferation, migration, and invasion by PAR-1 antagonist treatment. (a) Evaluation by scratch assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. Scale bar = 100 μm. Objective: 100×. (b) Relative migration rate for scratch assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. (c) Evaluation by transwell assay with Matrigel invasion for 72 h. Scale bar = 50 μm. Objective: 200×. (d) Relative invasion rate for transwell assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. (e) Clonogenic assay in U251 and U87. (f) Measurement of colony number after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. n = 5 independent replicates. ns: No significant, ^✶✶✶P < 0.001. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1.

Figure 5:

Inhibition of ADAM15-induced EMT by PAR-1 antagonist treatment. (a) Western blotting of E-cadherin, N-cadherin, and Vimentin protein expression after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment in U251. (b) Histogram of E-cadherin, N-cadherin, and Vimentin protein expression in U251. (c) Western blotting of E-cadherin, N-cadherin, and Vimentin protein expression after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment in U87. (d) Histogram of E-cadherin, N-cadherin, and Vimentin protein expression in U87. (e) Immunofluorescence of E-cadherin and Vimentin staining in U251 and U87. Scale bar = 20 μm. Objective: 400×. n = 5 independent replicates. ns: Not significant, ^✶✶P < 0.01. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1, Epithelial-mesenchymal transition.