Objective: To investigate the effects of p-fifty three inhibitor-alpha (PFT-alpha), a p53 inhibitor, on the proliferation and apoptosis of colon epithelial cells damaged by hyperthermic chemotherapy.
Methods: Normal epithelial cells were obtained from the mucosa at least 10 cm away from the cancer tissue in a specimen of large intestine cancer resected during operation and cultured. PFT-alpha at different concentrations was added into the culture fluid to observe its effects on the proliferation of the epithelial cells. Epithelial cell in logarithmic growth phase were inoculated in 6-well plate and divided into 3 groups: normal control (CON) group; hyperthermic chemotherapy (HTC) group, undergoing treatment of cisplatin and bath in water at 43 degrees C; and PFT-alpha + HTC group, undergoing treatment of PFT-alpha at different concentrations, cisplatin, and warm water bath. The cell apoptosis was observed by annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry (FCT). The cell cycle was observed by PI staining and FCT. Western blotting was used to detect the protein expression of cyclinB1 and Cdc2, and RT-PCR was used to detect the mRNA expression of cyclinB1.
Results: PFT-alpha at the concentration > 60 micromol/L significantly inhibited the proliferation of the large intestine epithelial cells. The natural apoptosis rate of the large intestine epithelial cells (CON group) was 2.9% +/- 0.4%, the apoptosis rate was 27.0% +/- 2.1% in the HTC group, and the apoptosis rates of the PFT-alpha + HTC group were 14.8% +/- 1.5%, 9.7% +/- 1.2%, 6.1% +/- 1.3%, and 3.8% +/- 0.3%, on a downward trend, corresponding to the increase of PFT-alpha concentration from 0, 20, 30, to 40 micromol/L (all P < 0.05). The G(0)/G(1) phase rate of epithelial cells was higher and the S phase rate was lower significantly in the PFT-alpha + HTC group. The G(2)/M phase rate was higher since the PFT-alpha concentration reached 10 micromol/L and then increased along with the increase of the PFT-alpha concentration; the S phase rates of the PFT-alpha + HTC group with different PFT-alpha concentrations were all significantly higher than that of the HTC group (all P < 0.01), however, were still lower than that of the CON group (all P < 0.01). The protein expressions of cyclinB1 and Cdc2 in the PFT-alpha + HTC group were both significantly higher than those in the CON and HTC groups (all P < 0.01), without a significant difference between the latter 2 groups. The mRNA expression of cyclinB1 in the PFT-alpha + HTC group increased along with the increase of the PFT-alpha concentration, and there wee significant differences in the mRNA expression of cyclinB1 between the CON and PFT groups and PFT-alpha + HTC group with the PFT-alpha concentration > or = 10 micromol/L (P < 0.05 or P < 0.01).
Conclusion: PFT-alpha dose-dependently protects the hyperthermic chemotherapy-induced damage to the large intestine epithelial cells via upregulation of protein and mRNA expression of cyclinB1, increasing the phosphorylation level of Cdc2, decreasing the cyclinB1/Cdc2 activity, and increasing the G(2)/M phase rate of the cells.