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To further examine the eﬀects of 8-AHN on cell proliferation and survival, HCT116 cells were treated with 5 μM 8-AHN at 0, 24, 48 h. FCM showed that the percentage of cells at the G2/M phase sig-nificantly increased from 29.2% to 53.1% or 57.9% after treatment with 5 μM 8-AHN for 24 or 48 h, respectively (Fig. 2A). Consistently, the expression of cyclin A and cyclin B, the key regulators of G2 to M phase progression, decreased in HCT116 and LOVO at a dose-depen-dent manner (Fig. 2B). As well, real-time quantitative PCR showed an mRNA expression decrease in cyclin B and cyclin A within 24 h treat-ment with 8-AHN in a dose-dependent manner (Fig. 2C). These data suggested that 8-AHN induced a cell cycle arrest at the G2/M phase through down-regulation of cyclin B and cyclin A.
Fig. 6. 8-AHN inhibited colorectal cancer tumor xenograft growth in vivo.
(A) Tumor volumes in 8-AHN-treated groups and the control groups were shown.
(B) Tumor weights data analysis were shown. (C) Body weight was measured at the indicated time intervals. (D) Tumor size was measured at the indicated time intervals.
(E) Tumor specimens were subjected to im-munohistochemical staining with HG-9-91-01 specific to BAX, PCNA, p53 and cyclin B.
(F) Representative images of H&E staining in paraﬃn sections of lung and liver were shown. Data were presented as means ± S.D. from three independent experiments.
3.3. 8-AHN induced cell apoptosis
According to the cell cycle analysis above, we suppose that 8-AHN might induce the apoptosis of colorectal cancer cells. To confirm this, HCT116 cells were treated with 5 μM of 8-AHN for 24 h or 48 h, then, the cells was harvested and stained with Annexin V-FITC and propi-dium iodide (PI), and analyzed by flow cytometry. As shown in Fig. 3A, when the cells were incubated with 8-AHN at 5 μM for 0 h, 24 h and 48 h, the percentages of early and late apoptosis cells were 2.35%, 21.49%, 31.57%. The apoptotic outcome was further corroborated by showing a significant increase protein levels in cleaved caspase-3 at 8 μM by western blot (Fig. 3B).
3.4. 8-AHN enhanced p53 expression and transcriptional activity
As a stress-activated transcription factor, p53 plays a very important role in cell cycle arrest and apoptosis. Thus, we examined whether p53 was functionally activated in HCT116 cells in response to 8-AHN. We first determined the expression characteristics of p53 in 8-AHN-treated HCT116 cells. As shown in Fig. 4A, treatment with 8-AHN for 24 h in-creased the p53 protein levels via a concentration-dependent manner. Real-time quantitative PCR also showed an increase in p53 mRNA ex-pression within 24 h of treatment with 8-AHN (Fig. 4B). These data suggested that 8-AHN could regulate the expression of p53 in cancer cells.
Furthermore, we investigated whether the up-regulation of p53 was associated with an increased transcriptional activity. Indeed, we found that, significant increases in p21, BAX and FAS expression were ob-served at the transcriptional and protein levels by 8-AHN treatment as well (Fig. 4A, B and C). These data demonstrated that 8-AHN enhanced p53 expression and transcriptional activity.
3.5. Inhibition of p53 attenuated 8-AHN suppressed cells viability
To determine that p53 exists an essential role in response to 8-AHN treatment, HCT116 cells were pre-treated with the p53 inhibitor PFT-α before treatment of 8-AHN. Western blot analysis showed that PFT-α can significantly inhibited the induction of p21, BAX, and FAS by 8-AHN treatment and also increased the expression of cyclin B (Fig. 5A). Consistently, FCM showed that silencing of p53 by PFT-α partially rescued cells from 8-AHN-induced apoptosis and cell cycle arrest. This is evident from the decrease in proportion of cells in sub-G1 and G2/M phase in PFT-α transfected HCT116 cells compared with control cells (Fig. 5B).