P2Y12 receptor antagonism inhibits proliferation, migration and leads to autophagy of glioblastoma cells

Abstract

Glioblastoma (GBM) is the most aggressive and lethal among the primary brain tumors, with a low survival rate and resistance to radio and chemotherapy. The P2Y₁₂ is an adenosine diphosphate (ADP) purinergic chemoreceptor, found mainly in platelets. In cancer cells, its activation has been described to induce proliferation and metastasis. Bearing in mind the need to find new treatments for GBM, this study aimed to investigate the role of the P2Y₁₂R in the proliferation and migration of GBM cells, as well as to evaluate the expression of this receptor in patients' data obtained from the TCGA data bank. Here, we used the P2Y₁₂R antagonist, ticagrelor, which belongs to the antiplatelet agent's class. The different GBM cells (cell line and patient-derived cells) were treated with ticagrelor, with the agonist, ADP, or both, and the effects on cell proliferation, colony formation, ADP hydrolysis, cell cycle and death, migration, and cell adhesion were analyzed. The results showed that ticagrelor decreased the viability and the proliferation of GBM cells. P2Y₁₂R antagonism also reduced colony formation and migration potentials, with alterations on the expression of metalloproteinases, and induced autophagy in GBM cells. Changes were observed at the cell cycle level, and only the U251 cell line showed a significant reduction in the ADP hydrolysis profile. TCGA data analysis showed a higher expression of P2Y₁₂R in gliomas samples when compared to the other tumors. These data demonstrate the importance of the P2Y₁₂ receptor in gliomas development and reinforce its potential as a pharmacological target for glioma treatment.

Keywords: ADP; Glioblastoma; P2Y12 receptor; Purinergic system; Ticagrelor.

Fig. 1

Cell viability and proliferation of GBM cells. Effect of the P2Y₁ antagonist MRS2179 (5–50 µM for 24 h) and the P2Y₁₂ antagonist ticagrelor (5–200 µM for 24 h) were assessed using MTT assay for A and C U251 and B and D patient-derived GBM cells (LS12). For cell count, E U251 and F LS12 cells were treated with ticagrelor (50 μM) and/or ADP (100 μM) for 24 h. The cell number was expressed in % in relation to the control. Dimethylsulfoxide (DMSO) was used as a vehicle control. Values represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate evaluated by one-way ANOVA followed by post hoc Tukey. *Means difference from the control group and # means difference from the ADP group. ^*,#p < 0.05, ^**,##p < 0.01, and.^***,###p < 0.001

Fig. 2

Colony formation assay and ADP hydrolysis activity. Glioma cells A U251 and B LS12 were treated with ticagrelor (50 μM) and/or ADP (100 μM) for 24 h. In C, representative image of colony formation. The number of colonies is expressed as an absolute number. The ADP hydrolysis activity was measured using the malachite green method in D U251 and E LS12 cells treated with ticagrelor (50 μM) for 24 h. Values are represented as specific enzyme activity in nanomoles of inorganic phosphate per minute per milligram of protein. In (F), the comparison of basal hydrolysis between the GBM cell lines. Dimethylsulfoxide (DMSO) was used as a vehicle control. Values represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate evaluated by one-way ANOVA followed by post hoc Tukey. *Means difference from the control group and # means difference from the ADP group. ^*,#p < 0.05, ^**,##p < 0.01, and.^***,###p < 0.001

Fig. 3

Nuclear morphology analysis (NMA). U251 and LS12 cells were treated with ticagrelor (50 µM) for 24 h. In (A), the results are presented graphically and in N, normal nuclei; I, irregular core; LR, large and regular core; LI, large irregular core; SR, small, regular nucleus; S, small core; SI, small irregular nucleus (B) representative image

Fig. 4

Cell cycle and death analysis. For cell cycle analysis, A U251 and B LS12 were treated with ticagrelor (50 μM) for 24 and C U251 and D LS12 were treated with ticagrelor (50 μM) for 72 h. Cell cycle was analyzed by flow cytometry with propidium iodide labeling. Cell death was assessed with an annexin V kit using flow cytometry in E U251 and F LS12 treated with ticagrelor (50 µM) for 24 h. Dimethylsulfoxide (DMSO) was used as a vehicle control. Values represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate evaluated by one-way ANOVA followed by post hoc Tukey. *p < 0.05, **p < 0.01,.^***p < 0.001

Fig. 5

Autophagy analysis. For authophagy analysis, A U251 and D LS12 cells were treated with ticagrelor (50 μM) for 24 h followed by acridine orange assay by flow cytometry. In B and C U251 and E and F LS12 representative SSC x FSC cytometry histograms of control and ticagrelor respectively. Dimethylsulfoxide (DMSO) was used as a vehicle control. Values represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate evaluated by one-way ANOVA followed by post hoc Tukey. *p < 0.05, **p < 0.01, ^***p < 0.001

Fig. 6

Migration and adhesion profile of GBM cells. In the wound healing assay, A U251 cells were treated with ticagrelor (50 μM) and/or ADP (100 μM) for 24 h. Then, the cells were photographed at 0 h and 24 h and the cell migration space between the edges was measured. In (B), representative images of the cell migration process. For spheroid migration test, LS12 spheroids (C) were treated with ticagrelor (20 μM) and/or ADP (20 μM) for 48 h. Afterwards, they were photographed at 0 h and 72 h and the extent of migration was measured. In (D), representative image of the experiment. For the adherence assay, the cells E U251 and F LS12 cells were treated with ticagrelor (50 μM) and/or ADP (100 μM) and were seeded to adhere for 1 h. qPCR assay for metalloproteinases 2 and 9 was performed in G U251 and H LS12 cells treated with ticagrelor (50 μM) and the difference between the control group and the treated group was quantified. Dimethylsulfoxide (DMSO) was used as a vehicle control. Values represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate analyzed by Student t-test. *Means difference from the control group and #means difference from the ADP group. ^*,#p < 0.05, ^**,##p < 0.01, and.^***,###p < 0.001