Cancer Biology Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave., 430030, Wuhan, Hubei, People's Republic of China.
J Cancer Res Clin Oncol. 2010 Mar;136(3):447-56. doi: 10.1007/s00432-009-0675-4. Epub 2009 Sep 16.
Chemoresistance severely restricts the anti-cancer medicines from effectively treating human ovarian cancer, which has been shown to develop and survive in the specific hypoxic environments. To understand the relationship between hypoxia and chemoresistance, we investigated the potential role of hypoxia in the pathophysiology of chemoresistance, especially focusing on hypoxia-inducible factor 1alpha (HIF-1alpha).
The A2780 ovarian cancer cells are cultured in gradient hypoxic conditions (5% O(2), 3% O(2), and 1% O(2)), the sensitivity of the cells to paclitaxel and the cell inhibitory rate were determined by MTT assay. The expression and the transcriptional activity of HIF-1alpha were examined by western blot, Immunocytochemical staining, reverse transcription-polymerase chain reaction (RT-PCR), and the dual luciferase reporter system, respectively. The cell cycle distribution was analyzed by flow cytometry. In addition, we silence HIF-1alpha expression by performing RNA interference.
MTT assay demonstrates that hypoxic challenge substantially reduces the susceptibility of cells to paclitaxel at all the tested concentrations. Coincident with this is the activation of HIF-1alpha in nuclear, which displays the increased transcriptional activity and high protein expression. Hypoxic manipulation (5% O(2), approximately 1% O(2)) significantly increased the cell population at G0/G1. Interestingly, knockdown of endogenous HIF-1alpha significantly alleviates the chemoresistance and promotes G1/S transition with the increased sensitivity of A2780 cells to paclitaxel under each hypoxic condition.
It suggests that HIF-1alpha, stimulated by hypoxia, exerts a pivotal role in chemoresistance by G0/G1 arrest. Eliminating hypoxic conditions or silencing HIF-1alpha by siRNA might provide a potent tool to enhance paclitaxel effectiveness in treatment of human ovarian cancer.
化疗耐药严重限制了抗癌药物有效治疗人类卵巢癌，而卵巢癌已被证明在特定的低氧环境中发展和存活。为了了解低氧与化疗耐药之间的关系，我们研究了低氧在化疗耐药病理生理学中的潜在作用，特别是重点关注缺氧诱导因子 1α（HIF-1α）。
将 A2780 卵巢癌细胞在梯度低氧条件（5% O₂、3% O₂ 和 1% O₂）下培养，通过 MTT 法测定细胞对紫杉醇的敏感性和细胞抑制率。通过 Western blot、免疫细胞化学染色、逆转录-聚合酶链反应（RT-PCR）和双荧光素酶报告系统分别检测 HIF-1α的表达和转录活性。通过流式细胞术分析细胞周期分布。此外，通过 RNA 干扰沉默 HIF-1α表达。
MTT 法表明，低氧刺激在所有测试浓度下均显著降低细胞对紫杉醇的敏感性。同时，HIF-1α在核内被激活，表现出转录活性增加和高蛋白表达。低氧处理（5% O₂，约 1% O₂）显著增加了 G0/G1 期的细胞群体。有趣的是，内源性 HIF-1α 的敲低显著减轻了耐药性，并在每种低氧条件下增加了 A2780 细胞对紫杉醇的敏感性，促进了 G1/S 期的转变。
这表明，低氧刺激的 HIF-1α 通过 G0/G1 期阻滞在化疗耐药中发挥关键作用。消除低氧条件或通过 siRNA 沉默 HIF-1α可能为增强紫杉醇在治疗人类卵巢癌中的有效性提供有力工具。
