Antiplatelet Drug Ticagrelor Enhances Chemotherapeutic Efficacy by Targeting the Novel P2Y12-AKT Pathway in Pancreatic Cancer Cells

Figures

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  Figure 1. The P2Y12 receptor, activated by ADP, triggers AKT activation in PDAC cells. (A) Immunoblots show the expression of P2Y12 and EGFR in PDAC cells (AsPC-1, BxPC-3, MiaPaCa-2, CFPAC-1 and PANC-1) and the normal pancreatic duct cells h-TERT-HPNE. Platelets were used as a positive control for P2Y12. Cells were seeded at 3 × 105 cells/well in a 6-well plate for 24 h, then washed, lysed and the proteins were collected and quantified. (B,C) Relative P2Y12 and EGFR expression in five PDAC cell lines and h-TERT-HPNE cells. The expression level was quantified and normalised to the loading control, α-actinin, with automated software Image Lab (version 5.1, BioRad, Hercules CA, USA) and represented as columns using GraphPad Prism 8 (GraphPad Software, Inc, CA, USA). (D) The P2Y12 inhibitor, ticagrelor, reduced ADP-induced AKT activation in AsPC-1 and BxPC-3. Briefly, cancer cells were seeded in a 12-well plate and after 24 h, cells were starved for 6 h, then treated with ticagrelor (5 µM) combined with ADP (100 µM) and the cells were further incubated for 30 min in serum-free media. The figure shows a representative blot from three independent experiments. (E) Extracellular ADP release from…

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  Figure 2. P2Y12 regulates EGFR activation, SLUG and ZEB1 expression and enhances PDAC cells viability. (A) Immunoblots showing the expression of P2Y12, p-EGFR Y1068, EGFR, SLUG and ZEB1 in AsPC-1 and BxPC-3 after P2Y12-specific siRNA treatment. Cancer cells were seeded and treated with P2Y12 siRNA or a negative control siRNA, as described in the Methods and Materials section. (B) The columns represent the fold change of protein levels (p-EGFR Y1068, SLUG and ZEB1) relative to the negative control siRNA-treated cells, normalised to 1 (n ≥ 3). Statistical analysis was performed using one sample t-test (GraphPad Prism 8) comparing the mean of siP2Y12 treatment with the normalised value 1. (C) Cell viability of AsPC-1 and BxPC-3 cells following knockdown of P2Y12 compared with the negative control siRNA-treated cells (n ≥ 3), calculated using one sample t-test (Graphpad Prism 8) and the normalised control group mean of 100%. Data are presented as mean ± SEM. *** p < 0.001, ** p < 0.002, * p < 0.033. siNeg: siRNA negative control.

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  Figure 3. Ticagrelor attenuated EGF-stimulated AKT activation and potentiated the anticancer activity of erlotinib. (A) Immunoblots showing the expression of p-EGFR Y1068, p-AKT S473 and p-ERK1/2 in serum-starved AsPC-1 and BxPC-3 cells treated with EGF (10 ng/mL) combined with ticagrelor (5 µM) or apyrase (5 U/mL) for 1 h. (B) Immunoblots showing the expression of p-EGFR Y1068, p-AKT S473 and p-ERK1/2 in serum-starved AsPC-1 and BxPC-3 cells treated with ADP (100 µM) combined with erlotinib (5 µM) for 30 min. EGF +/− erlotinib was used as a control for EGFR activation. (C) Cell viability of AsPC-1 and BxPC-3 following treatment with erlotinib (0.001–10 µM) with and without ticagrelor (2.5 µM) for 72 h. Data are presented as mean ± SEM. Two-way ANOVA with post-hoc Bonferroni’s multiple comparison test (GraphPad PRISM 8.0) was used to examine the significance of the mean; n = 5, *** p < 0.001. Table 1A,B show the combination index (CI) values calculated using Chou–Talalay’s method and can be interpreted as follow: CI < 0.9, synergism, CI > 1.1, antagonism, CI = 0.9–1.1, additive. (D) Immunoblots show the expression of p-EGFR Y1068, p-AKT S473 and p-ERK1/2 in non-starved AsPC-1 and BxPC-3…

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  Figure 4. Ticagrelor treatment reduced PDAC cell viability and enhanced apoptosis through attenuating AKT activation in PDAC cells. (A) Relative cell viability in AsPC-1, BxPC-3, MiaPaCa-2, PANC-1, CFPAC-1 and h-TERT-HPNE cells upon treatment with ticagrelor (0–20 µM) for 72 h (n ≥ 3). Table 2 shows the IC50 (µM) of ticagrelor calculated using GraphPad PRISM 8 and log (inhibitor concentration) versus the normalised response (variable slope). (B) AsPC-1 and BxPC-3 cells were treated with ticagrelor (0–10 µM) for 12 h and analysed for apoptosis (caspase 3/7 activation). The columns represent the fold change in the level of activated caspase 3/7 relative to the control vehicle-treated cells, measured as described in the Methods and Materials section. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett’s multiple comparison test was used to examine the significance of the mean; n ≥ 4, *** p < 0.001, ** p < 0.002, * p < 0.033. (C) Detection of caspase 3 activity in live cells treated with vehicle or ticagrelor 10 µM for 12 h. The caspase 3 substrate, once cleaved by caspase 3, formed a DNA dye, which stained the nucleus bright green. (D) Immunoblots showing the expression of…

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  Figure 5. Ticagrelor synergised with chemotherapy in PDAC cells in vitro. (A–C) Relative cell viability in AsPC-1, BxPC-3 and MiaPaCa-2 upon treatment with gemcitabine (0–100 µM), paclitaxel (0–100 nM) and cisplatin (0–100 µM) as single agents or in combination with ticagrelor (2.5 µM) for 72 h. The significance of the difference between viability of cells treated with chemotherapy alone or in combination with ticagrelor was tested using two-way ANOVA with post-hoc Bonferroni’s multiple comparison test; n ≥ 3, *** p < 0.0001, ** p < 0.001. The tables below the graphs show the CI values calculated using Chou–Talalay’s method and can be interpreted as follow: CI < 0.9, synergism, CI > 1.1, antagonism, CI = 0.9–1.1, additive.

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  Figure 6. Combined treatment of ticagrelor (Tica) and gemcitabine (GEM) reduced tumour growth in vivo. (A) Effect of different treatments on tumour weight and volume in each NOD/SCID mouse bearing BxPC-3 tumours. The significance of the difference between tumour weight and volume in mice bearing BxPC-3 tumours treated with a vehicle control or gemcitabine, ticagrelor or gemcitabine plus ticagrelor was tested using ordinary one-way ANOVA (weight) or two-way ANOVA (volume) with post-hoc Dunnett’s multiple comparison test; n = 8 for vehicle, n = 8 for gemcitabine, n = 7 for ticagrelor, n = 7 for gemcitabine plus ticagrelor. The significance of the difference between the tumour volumes was calculated using the two-way ANOVA Dunnett’s multiple comparison test to examine the significance of the mean. *** p < 0.001, * p < 0.033. (B) The level of plasma TGF-β1 in NOD/SCID mice without tumours, or age-matched NOD/SCID mice with BxPC-3 tumours treated with vehicle, gemcitabine, ticagrelor or ticagrelor plus gemcitabine (n = 4). The significance of the difference between the TGF-β1 level was calculated using the two-way ANOVA Dunnett’s multiple comparison test to examine the significance of the mean.…

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  Figure 7. Ticagrelor potentiated gemcitabine activity in the syngeneic tumour mouse model. Effect of different treatments on tumour weight and volume in a syngeneic pancreatic cancer mouse model (C57BL6/J mice transplanted with MT4-2D). The significance of the difference between the tumour weight and volume in a syngeneic pancreatic cancer mouse model treated with vehicle control or gemcitabine, ticagrelor or gemcitabine plus ticagrelor was tested using ordinary one-way ANOVA (weight) or two-way ANOVA (volume) with post-hoc Dunnett’s multiple comparison test; n = 4 for vehicle, n = 4 for gemcitabine, n = 5 for ticagrelor, n = 5 for gemcitabine plus ticagrelor. The significance of the difference between the tumour volumes was calculated using two-way ANOVA Dunnett’s multiple comparison test to examine the significance of the mean. * p < 0.033.