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. 2010 Mar;136(3):447-56.
doi: 10.1007/s00432-009-0675-4. Epub 2009 Sep 16.

Hypoxia induced paclitaxel resistance in human ovarian cancers via hypoxia-inducible factor 1alpha (V体育安卓版)

Lei Huang  1 , Qilin Ao, Qinghua Zhang, Xiaokui Yang, Hui Xing, Fang Li, Gang Chen, Jianfeng Zhou, Shixuan Wang, Gang Xu, Li Meng, Yunping Lu, Ding Ma
Affiliations

Hypoxia induced paclitaxel resistance in human ovarian cancers via hypoxia-inducible factor 1alpha

Lei Huang et al. J Cancer Res Clin Oncol. 2010 Mar.

Abstract

Purpose: 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) VSports手机版. .

Methods: 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 V体育安卓版. 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. .

Results: 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. V体育ios版.

Conclusions: It suggests that HIF-1alpha, stimulated by hypoxia, exerts a pivotal role in chemoresistance by G0/G1 arrest VSports最新版本. Eliminating hypoxic conditions or silencing HIF-1alpha by siRNA might provide a potent tool to enhance paclitaxel effectiveness in treatment of human ovarian cancer. .

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Figures

Fig. 1
Fig. 1
Hypoxia-induced HIF-1α expression associated with decreased susceptibility to Paclitaxel in ovarian cancer cells. The cells were treated with the concentrations of paclitaxel (0.2, 2, 20, and 200 μM) for 24 h under normoxic (21% O2) or hypoxic conditions (5% O2, 3% O2 and 1% O2). a Comparison of the inhibition rates of A2780 cells viability cultured in normoxia or in gradient hypoxia. MTT analyses present the X ± SE of three independent experiments. When treated with paclitaxel, hypoxia cells showed significantly lower cell inhibitive rate than the normoxia cells. (*P = 0.008, Fig 1a). b Cells were collected, the total protein were subjected to Western blot analysis with HIF-1α-specific antibody. β-actin was used to demonstrate equal loading. c HIF-1α expression of A2780 in normoxia or in gradient hypoxia. RNA samples were isolated from cells and were electrophoresed on 1% agarose gel stained with ethidium bromide for RT–PCR analysis. All PCRs were determined and co-amplified with GADPH as an internal control
Fig. 2
Fig. 2
Determination of HIF-1α activity in A2780 and A2780/siHIF-1α cells cultured in normoxia or hypoxia. A2780 cells and A2780/siHIF-1α cells were treated with 20-μM paclitaxel and incubated in normoxia or gradient hypoxia (5% O2, 3% O2 and 1% O2 oxygen) for 24 h. The pGL3-promoter-HRE was constructed as described in “Materials and Methods”. The luciferase activity was measured with a luminometer using the dual luciferase reporter system. Values and error bars shown in this graph represent the averages and standard deviations, respectively, of three independent experiments. Compared with normoxia A2780 cells *P < 0.05, compared with corresponding hypoxia cells **P < 0.05
Fig. 3
Fig. 3
The location of HIF-1α by immunocytochemical staining with HIF-1α antibody. a Cells cultured in normoxia for 24 h displayed the faint staining in the cytoplasm without the nuclear staining (black arrow). b Cells cultured in 5% O2, c in 3% O2, d in 1% O2 for 24 h showed strong nuclear staining (black arrows)
Fig. 4
Fig. 4
Down-regulation of HIF-1α by HIF-1α/siRNA transfection. siRNA pSIREN-DNR-DsRed express vector was used to transiently transfect HIF-1α/siRNA into A2780 cells, termed A2780/siHIF-1α. A2780 cells and A2780/siHIF-1α cells were exposed to 20 μM paclitaxel and incubated in normoxia or gradient hypoxia (5% O2, 3% O2 and 1% O2) for 24 h. a RNA samples were isolated from A2780 cells or from A2780/siHIF-1α cells. The RNA products were electrophoresed on 1% agarose gel stained with ethidium bromide for RT–PCR analysis. All PCRs were determined and co-amplified with GADPH as an internal control. b Cells were collected, the total protein were subjected to Western blot analysis with HIF-1α-specific antibody. β-actin was used as a loading control
Fig. 5
Fig. 5
HIF-1α/siRNA transfection reversed hypoxia-induced resistance to paclitaxel. A2780 and A2780/siHIF-1α cells were treated with paclitaxel of 0.2, 2, 20, and 200 μM for 24 h under normoxic or hypoxic conditions (5% O2, 3% O2 and 1% O2). Comparison of the inhibition rates of A2780 cells viability cultured. The inhibitive rate of A2780/siHIF-1α cells were significantly higher than those of A2780 cells in gradient hypoxia conditions (5% O2, 3% O2 and 1% O2), but there were no significant difference between A2780/siHIF-1α cells and A2780 cells in normoxia conditiaons. a In hypoxic condition containing 5% O2 (*P = 0.0032); b in hypoxic condition containing 3% O2 (*P = 0.0048); c in hypoxic condition containing 1%O2. (*P = 0.0061); d in normoxic condition (*P = 0.073). MTT analyses present the X ± SE of three independent experiments
Fig. 6
Fig. 6
Hypoxia induced G0/G1 Cell Cycle arrest in A2780 cells. A2780 and A2780/siHIF-1α cells were incubated in normoxia or gradient hypoxia (5% O2, 3% O2 and 1% O2) for 24 h. Cell were stained with 50 μg/ml of presidium iodide and analyzed by flow cytometry. a Cell cycle analysis and the DNA content of A2780 cells; b Cell cycle analysis and the DNA content of A2780/siHIF-1α cells; c G0/G1 phase cells ratios in A2780 cells and A2780/siHIF-1α cells incubated in normoxia or gradient hypoxia for 5% O2, 3% O2 and 1% O2 for 24 h (*P = 0.036; **P = 0.028; ***P = 0.031)
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