Zhang An-ping, Liu Bao-hua, Zhang Lian-yang, Tong Wei-dong, He Yu-jun, Fu Tao, Luo Dong-Lin
Department of General Surgery, Research Institute of Field Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Zhonghua Yi Xue Za Zhi. 2006 Jan 10;86(2):93-7.
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.
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.
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).
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.
探讨p53抑制剂p-53抑制剂-α（PFT-α）对热化疗损伤的结肠上皮细胞增殖和凋亡的影响。
从手术切除的大肠癌标本中距癌组织至少10 cm处的黏膜获取正常上皮细胞并进行培养。向培养液中加入不同浓度的PFT-α，观察其对上皮细胞增殖的影响。将对数生长期的上皮细胞接种于6孔板，分为3组：正常对照组（CON组）；热化疗（HTC）组，接受顺铂治疗并在43℃水浴；PFT-α + HTC组，接受不同浓度的PFT-α、顺铂和温水浴治疗。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（PI）染色和流式细胞术（FCT）观察细胞凋亡情况。采用PI染色和FCT观察细胞周期。采用蛋白质印迹法检测细胞周期蛋白B1（cyclinB1）和细胞分裂周期蛋白2（Cdc2）的蛋白表达，采用逆转录-聚合酶链反应（RT-PCR）检测cyclinB1的mRNA表达。
浓度>60 μmol/L的PFT-α显著抑制大肠上皮细胞的增殖。大肠上皮细胞（CON组）的自然凋亡率为2.9%±0.4%，HTC组的凋亡率为27.0%±2.1%，PFT-α + HTC组的凋亡率分别为14.8%±1.5%、9.7%±1.2%、6.1%±1.3%和3.8%±0.3%，呈下降趋势，对应PFT-α浓度从0、20、30至40 μmol/L升高（均P<0.05）。PFT-α + HTC组上皮细胞的G(0)/G(1)期比例较高，S期比例显著较低。当PFT-α浓度达到10 μmol/L时，G(2)/M期比例升高，随后随PFT-α浓度升高而增加；不同PFT-α浓度的PFT-α + HTC组的S期比例均显著高于HTC组（均P<0.01），但仍低于CON组（均P<0.01）。PFT-α + HTC组中cyclinB1和Cdc2的蛋白表达均显著高于CON组和HTC组（均P<0.01），后两组之间无显著差异。PFT-α + HTC组中cyclinB1的mRNA表达随PFT-α浓度升高而增加，PFT-α浓度≥10 μmol/L的CON组与PFT组及PFT-α + HTC组之间cyclinB1的mRNA表达存在显著差异（P<0.05或P<0.01）。
PFT-α通过上调cyclinB1的蛋白和mRNA表达、增加Cdc2的磷酸化水平、降低cyclinB1/Cdc2活性以及增加细胞的G(2)/M期比例，剂量依赖性地保护热化疗对大肠上皮细胞的损伤。
