[缺氧对人卵巢癌细胞对紫杉醇化疗敏感性的影响及其机制]

[Effect of hypoxia on the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism].

作者信息

Huang Lei, Zhang Qing-Hua, Ao Qi-Lin, Xing Hui, Lu Yun-Ping, Ma Ding

机构信息

Department of Obstetrics & Gynecology, Central Hospital of Wuhan, Wuhan 430014, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2007 Feb;29(2):96-100.

PMID:17645840

Abstract

OBJECTIVE

To explore the effect of hypoxia and hypoxia-inducible factor-1alpha (HIF-1alpha) on the expression of multidrug resistance gene-1 (mdr-1) and its coded p-glyeoprotein (P-gp) as well as the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism.

METHODS

The mRNA and protein levels of HIF-1alpha, mdr-1 and p-gp were studied by immunocytochemistry, semiquantitative reverse transcription-ploymerase chain reaction (RT-PCR) and Western blot analysis in human ovarian cancer cells (A2780) in 5% CO2 + 1% O2 hypoxic culture and 21% O2 normoxic culture, respectively. Methyl thiazolyl tetrazolium (MIT) was used to evaluate the chemotherapeutic sensitivity of A2780 cells to paclitaxel by inhibition rate. RNA interference technique was used and small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed as pSiHIF-1alpha, and transfected into A2780 cells. RT-PCR and Western blot were used to detect gene silencing effect on HIF-1alpha, the expressions of mdr-1 and p-gp. The inhibition rate was observed after HIF-1alpha gene silence.

RESULTS

HIF-1alpha at both mRNA and protein levels was induced significantly under hypoxia. The HIF-1alpha expression at mRNA level was oxygen gradient-independent, while HIF-1alpha expression at protein level was oxygen gradient-dependent. The inhibition rate of paclitaxel to hypoxic A2780 cells in 5% CO2 + 1% O2 was significantly lower than that in normoxic A2780 cells (P <0.05). The shRNAs plasmid targeting HIF-1alpha was constructed successfully and HIF-1alpha gene was silenced in A2780 cells efficiently followed by mdr-1 and p-gp down-regulation. The inhibition rate was greatly increased in hypoxic A2780/siHIF-1alpha cells.

CONCLUSION

Hypoxia can decrease the chemotherapeutic sensitivity of human ovarian cancer A2780 cells to paclitaxel through HIF-1alpha regulating the expression of mdr-1 and p-gp.

摘要

目的

探讨缺氧及缺氧诱导因子-1α（HIF-1α）对多药耐药基因-1（mdr-1）及其编码的P-糖蛋白（P-gp）表达的影响，以及对人卵巢癌细胞紫杉醇化疗敏感性的影响及其机制。

方法

分别采用免疫细胞化学、半定量逆转录-聚合酶链反应（RT-PCR）及蛋白质免疫印迹法检测人卵巢癌细胞（A2780）在5%二氧化碳+1%氧气低氧培养及21%氧气常氧培养条件下HIF-1α、mdr-1及P-gp的mRNA和蛋白水平。采用噻唑蓝（MTT）比色法通过抑制率评价A2780细胞对紫杉醇的化疗敏感性。运用RNA干扰技术，构建靶向HIF-1α的小发夹RNA（shRNAs）真核表达载体pSiHIF-1α，并转染至A2780细胞。采用RT-PCR及蛋白质免疫印迹法检测对HIF-1α的基因沉默效果，以及mdr-1和P-gp的表达。观察HIF-1α基因沉默后的抑制率。

结果

低氧状态下HIF-1α的mRNA和蛋白水平均显著上调。HIF-1α的mRNA水平表达与氧浓度梯度无关，而蛋白水平表达与氧浓度梯度有关。5%二氧化碳+1%氧气低氧培养的A2780细胞对紫杉醇的抑制率显著低于常氧培养的A2780细胞（P<0.05）。成功构建靶向HIF-1α的shRNAs质粒，A2780细胞中HIF-1α基因被有效沉默，随后mdr-1和P-gp表达下调。低氧培养的A2780/siHIF-1α细胞的抑制率显著升高。