低氧对胰腺癌细胞株PC-3中IAP-2表达的影响机制

Effect of hypoxia on apoptosis inhibitory protein 2 expression and its mechanism in pancreatic cancer cell line PC-3

目的:研究极度低氧下人胰腺癌细胞株PC-3中IAP-2表达的变化,初步探讨其与HIF-1的相关机制.方法:PC-3细胞株分组孵育:常氧组,体积分数为20mL/LO2/50mL/LCO2/930mL/LN2低氧组4h,950mL/LN2/50mL/LCO2极度低氧组1、3、5h,950mL/LN2/50mL/LCO2低氧3h后复氧,另取一组加入300μmol/L氯化钴常氧孵育.用细胞免疫化学定性检测IAP-2蛋白表达;蛋白裂解液提取胞质胞核蛋白,用Westernblot检测IAP-2蛋白水平变化,同时对比HIF-1蛋白表达;用RT-PCR检测IAP-2基因水平改变.结果:免疫细胞化学检测IAP-2蛋白在PC-3细胞中呈阳性表达,定位于胞质.Westernblot显示常氧、低氧4h可检测到IAP-2蛋白,表达无差异,极度低氧1hIAP-2蛋白表达明显增加(t=3.300,P<0.05),3、5hIAP-2持续高表达,各时间段无显著性差异,复氧后恢复基线水平.实验还显示了HIF-1和IAP-2蛋白的表达差异:常氧组HIF-1未表达,低氧4h可诱发,IAP-2保持基线水平;极度低氧HIF-1无变化,IAP-2表达明显增高;复氧后HIF-1不表达,IAP-2恢复基线表达;加入氯化钴后HIF-1恢复表达,IAP-2保持基线,初步表明IAP-2蛋白表达上调有独立于HIF-1的作用机制.RT-PCR检测表明,极度低氧1、3、5h后IAP-2mRNA表达明显高于常氧组(t=6.900,P<0.05),各时间组无显著差异,复氧后恢复基线水平.该结果与上述IAP-2蛋白表达上调一致,初步表明IAP-2上调发生在转录水平.结论:极度低氧下IAP-2在人胰腺癌细胞株PC-3表达上调,并具有独立于HIF-1的抗凋亡机制.

AIM： To investigate the expression of apoptosis inhibitory protein 2 （IAP-2） in pancreatic cancer cell PC-3 under severe hypoxia, and to explore its relation with hypoxia inducible factor I（HIF-1）. METHODS： PC-3 cells were cultured under different conditions as follows： normoxia; 20 mL/L O2, 50 mL/L CO2, and 930 mL/L N2 for 4 h （hypoxia）; 950 mL/L N2 and 50 mL/L CO2 for 1, 3, and 5 h, respectively （severe hypoxia）; reoxygenation after 1 h of severe hypoxia; normoxia with colalt chloride （300 mol/L）. Immunocytochemistrywas used to qualitatively evaluate the expression of IAP-2 protein. After extraction of cytoplasmic and nuclear proteins, Western blot was used to quantitatively determine the expression of IAP-2 protein, which was compared with the expression of HIF-1 protein. Then the expression of IAP-2 mRNA was detected by reverse transcription- polymerase chain reaction （RT-PCR）. RESULTS： IAP-2 protein was positively expressed in the cytoplasm of PC-3 cells. There was no significant difference between the expression levels of IAP-2 protein under normoxia and hypoxia. The expression of IAP-2 protein was markedly increased （t = 3.300, P 〈0.05） 1 h after severe hypoxia and remained high at 3 or 5 h. There was no significant difference among different time points （P 〈0.05）. Reoxygenation led to basal expression of IAP-2 protein and mRNA. HIF-1 expression was undetectable in normoxic PC-3 cells, but it was induced by hypoxia. Under severe hypoxia, HIF-1 was modestly expressed, but IAP-2 was abundantly expressed. After reoxygenation, the expression of HIF-1 disappeared, and IAP-2 returned to the basal level. Colalt chloride activated HIF-1 but not IAP-2. One hour after severe hypoxia, the expression of IAP-2 mRNA was evidently higher than that under normoxia （t = 6.900, P 〈0.05） and remained high at 3. 5 h. There was no significant different among different time points （P 〉0.05）. CONCLUSION： Severe hypoxia induces the upregulation of IAP-2 in PC-3 cells through HIF-1- independent pathways.