Aspirin triggers TRAIL-induced apoptotic cell death via enhanced caspase activity 10

2. Aspirin triggers TRAIL-induced apoptotic cell death via enhanced caspase activity

To see the type of cell death, we performed annexin-V-FITC / PI double staining for the measurement of phosphatidylserine (PS) exposure. PI staining was also done for the measurement of sub-G1 population (Fig. 3 and Fig. 4). The annexin-V-FITC/PI staining for flow cytometry is the most commonly used method for detecting apoptosis. Annexin-V is a calcium-dependent phospholipid binding protein that has a high affinity for the PS, a plasma membrane phospholipid. One of the earliest features of apoptosis is the translocation of PS from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external environment. Annexin-V binds to PS exposed on the cell surface and identifies cells at an earlier stage of apoptosis than assays based on DNA fragmentation. The apoptotic cells with degraded DNA appear as cells with hypodiploid DNA content and are represented in so-called "sub-G1" peaks on DNA histograms.

Early apoptotic cells detected by annexin-V-FITC were only 0.5% and 5% in aspirin and TRAIL alone treatment, respectively. However, they presented 3.2%, 7.3%, or 11.0% when the cells were pre-treated with aspirin for 24hr and continuously treated with TRAIL for 4hr, 6hr, or 8hr (Fig.3D, E, F and G), indicating that aspirin promoted TRAIL–induced apoptotic cell death in a time-dependent manner. Percentage of sub-G1 population was low in the untreated control cells (6.1%) (Fig.4A). Percentage of sub-G1 population in TRAIL-treated cells (4.7%) (Fig. 4C) was significantly increased in co-treated cells with TRAIL and aspirin (43.3%) (Fig. 4D and F). Next, we examined whether apoptotic cell death induced by the combined treatment depends on caspase activities. First we performed western blotting analysis. The combination markedly induced the activation of caspase-8, -9, and -3 (Fig. 5).

To further confirm that caspase activation is required for the increased apoptosis in combination treated HeLa cells, a pan-caspase inhibitor zVAD-fmk (50uM) was added to the cells 1 hour before treatment with aspirin for 24 hours and TRAIL for 8 hours. They were analyzed by flow cytometry for annexin-V-FITC staining, and the microscope observation was also performed (Fig. 2, Fig. 3C, and Fig. 4E). The characters associated with apoptosis, such as cell morphology, exposure of PS, subG1 population and PARP cleavage, were all completely disappeared by a pan-caspase inhibitor, zVAD-fmk. These results strongly

- 11 -

suggest that aspirin promotes TRAIL-induced apoptotic cell death through enhancement of caspase activation.

- 12 -

Fig. 3. Aspirin(ASA) enhanced TRAIL-induced apoptosis. A.HeLa cells were untreated.

B. Aspirin was treated for 32hr. C. TRAIL was treated for 8hr. D~F. Aspirin was treated for 24hr and TRAIL was continuously treated for 4hr (D), 6hr (E) and 8hr (F). G. z-VAD was pretreated for 1hr and followed by treatment of aspirin and TRAIL for 24hr and 8hr, respectively. Concentrations of aspirin, TRAIL and z-VAD-fmk were 5mM, 100ng/ml and 50uM, respectively. HeLa cells were seeded at 2 х 10⁵ cells per well of 6-well culture plate and stabilized for 20hr before any treatment.

- 13 - A. B.

C. D. E.

Fig. 4. Pretreatment of aspirin enhanced TRAIL-induced apoptosis. A.Untreated cells. B.

Aspirin was treated for 32hr. C. TRAIL was treated for 8hr. D.E. Aspirin was treated for 24hr and followed by treatment of TRAIL for 8hr with (E) or without (D) pretreatment of 50uM zVAD-fmk for 1hr, respectively. HeLa cells were seeded at 2 х 10⁵ cells per 6-well plate and stabilized for 20hr. Concentrations of aspirin and TRAIL were 5mM and 100ng/ml, respectively.

- 14 -

Fig.5. The combination treatment of aspirin and TRAIL promoted caspase activity.

HeLa cells were seeded at 2 х 10⁵ cells per 6-well plate and stabilized for 20hr. Aspirin was treated for 24hr and followed by TRAIL treatment for 8hr. Concentrations of aspirin and TRAIL were 5mM and 100ng/ml, respectively. Cells were harvested and equal amount of protein was processed for western blot analysis to detect target proteins.

- 15 -

Fig. 6. Apoptotic cell death induced by the combination of aspirin and TRAIL was inhibited by z-VAD. HeLa cells were pretreated with 50uM of z-VAD for 1hr and aspirin and TRAIL were treated as previously described. Concentrations of aspirin and TRAIL were 5mM and 100ng/ml, respectively. Cells were harvested and equal amount of protein was processed for western blot analysis to detect target proteins.

- 16 -

3. Aspirin results in TRAIL-induced caspase-dependent mitochondrial membrane potential change and cytochrome c release

We observed the decrease of Bid and activation of caspase-9 in the combination treatment by western blotting (Fig. 5). These results could be expected that mitochondrial pathway was involved in the combination treatment-induced apoptotic cell death. To confirm this, we checked mitochondrial membrane potential (MMP) change and the release of cytochrome c to cytosol. Additionally, we investigated whether MMP change and the release of cytochrome c were dependent on caspase activities. HeLa cells were incubated with 5mM aspirin for 24hr and followed by 100ng/ml TRAIL treatment for 8hr and analyzed for MMP change using JC-1 dye. As shown in Fig.7D and F, value of FITC/PE was clearly increased in the combination treated cells, indicating that the combined treatment induced loss of MMP (∆Ψm). Furthermore, pre-incubation of zVAD-fmk blocked the loss of ∆Ψm induced by the combination treatment (Fig. 7E), suggesting MMP loss was caspase-dependent. To analyze clearly whether cytochrome c was released to cytosol from mitochondria and it was also caspase-dependent, western blotting was carried out with cytosolic fraction. Cytochrome c was only detected in the combination treatment (Fig. 8). Cytochrome c release can be either dependent or independent on caspase activity (Green, 1998). Therefore, we examined whether zVAD-fmk was capable of preventing the release of cytochrome c. As shown in Fig 8, cytochrome c in cytosolic fraction was completely disappeared by the pre-treatment of zVAD-fmk. These results suggest that the combined treatment of aspirin with TRAIL in HeLa cells promotes mitochondrial pathway of apoptosis depending on caspase activities.

- 17 -

Fig. 7. The combination of aspirin and TRAIL induced MMP change. A.Untreated cells.

B. 5mM aspirin was treated for 32hr. C. 100ng/ml TRAIL was treated for 8hr. D~E. Aspirin was treated for 24hr and followed by TRAIL treatment for 8hr with (D) or without (E) pretreatment of 50uM zVAD-fmk for 1hr. HeLa cells were seeded at 2 х 10⁵ cells per 6-well plate and stabilized for 20hr.

- 18 -

Fig. 8. A pan-caspase inhibitor, zVAD-fmk, prevented cytochrome c release from mitochondria of the combination treated cells. To investigate whether cytochrome c was released to cytosol from mitochondria and it was caspase-dependent, cytosol fraction was obtained for western blotting analysis. HeLa cells were treated with 5mM aspirin for 24hr followed by 100ng/ml TRAIL for 8hr in the presence or absence of pretreatment of 50uM zVAD-fmk for 1hr. HeLa cells were seeded at 1х10⁶ cells in 60 ф dish and stabilized for 20hr.

- 19 -

4. Aspirin makes TRAIL-resistant HeLa cells TRAIL-sensitive through inhibition of