br stages II The percentage of the cells in
stages, II: The percentage of the EX 527 in each stage. VI: viable cells; EA: early apoptosis; LA: late apoptosis; N: necrosis. (B) Caspase activity assay of 8-PN in HCT-116 cells. The luminescence analysis demonstrated a significant diﬀerence in expression level of caspases 3/7, 8 and 9 after 48 and 72 h. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ****p < 0.0001 indicate significant diﬀerences as compared to control.
dramatic increase in late apoptosis phase as compared to the untreated control cells (0%). This result suggests that apoptosis occurred in a time-dependent manner in 8-PN-treated cells.
3.5. Caspase activity
In this study, caspase activity of 8-PN was measured using the caspase Glo kit. Pro-caspase was used as substrate for luciferase activity in which its cleavage results in generation of chemilumiscence signal. It is to be noted that in this experiment, the intensity of luminescence produced by pro-caspase cleavage is proportional to the amount of
caspase activity of treated cells.
As shown in Fig. 3B, our results indicated that apoptosis was acti-vated through both intrinsic and extrinsic pathways following treat-ment with 8-PN at 48 h time point. Activation of caspase cascades was not significantly altered in the 8-PN-treated cells after 24 h as compared to the untreated control cells. However, after 48 h of treatment, the amount of caspase cascades increased significantly as compared to the control and this trend was observed until 72 h of treatment. Since caspase-8 and 9 activities had significantly increased in the 8-PN treated cells as compared to the control, this result indicates that both extrinsic and intrinsic pathways of apoptosis were activated after 48
Cancer development is usually characterized by abnormal cell pro-liferation. Mutation in any of the genes that are involved in the reg-ulation of cell cycle or growth factor signaling pathway can lead to cell signal autonomy . Our cell cycle results indicate that the anti-proliferative properties of 8-PN is due to the potential activity of 8-PN to arrest HCT-116 cells at G0/G1 phase of cell cycle. Allsopp et al.  have been reported that Caco-2 colon cancer cells treated with 8-PN were arrested at G0/G1 phase, which is similar to our findings, however for MCF-7 breast cancer cells, arrest in the presence of 8-PN occurred at S phase due to the inhibition of Cyclin D . The discrepancy in the findings between studies indicates that cell cycle arrest properties of 8-PN is cell specific and might vary from one cancer cells to another.
Beside the anti-proliferative properties of 8-PN, the eﬀect of this compound on induction of apoptosis was investigated. One of the initial signs of apoptosis in cells is translocation of phosphatidylserine (PS) from the inner face of the plasma membrane to the outside of plasma membrane. Consequently, construction of the cells will be lost and disruption in cell cycle will occur. Once PS appears on the surface, fluorescent annexin-V will be conjugated to the PS . This feature of the cells is used to measure the apoptosis of treated cancer cells. Apoptosis plays a remarkable role in regulating cell death and can be an important target in the treatment of numerous diseases, particularly cancer .
Our results demonstrate that 8-PN induces apoptosis through in-duction of extrinsic and intrinsic apoptotic pathways. In the presence of 8-PN caspase 8 and 9 which are the key factors for extrinsic and in-trinsic apoptotic pathways were activated time dependently. These findings confirmed the Annexin results, which showed that HCT-116 cells pass through the early and late apoptotic pathways and the number of the dead cells was increased in the presence of 8-PN time dependently. Moreover, AO/PI staining shows some morphological changes in 8-PN treated cells, which considered as features of apoptosis.