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HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-Glycoprotein

PLoS ONE (2014) 9(6)
DOI: 10.1371/journal.pone.0098882
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Abstract

Background: Multidrug resistance (MDR) is one of the major reasons chemotherapy-based treatments fail. Hypoxia is generally associated with tumor chemoresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) remains unclear. This study aims to explore the molecular mechanisms of reversing colon cancer MDR by focusing on the target gene HIF-1α. Methods: A chemotherapeutic sensitivity assay was used to observe the efficiency of MDR reversal in LoVo multicellular spheroids (MCS). The apoptotic level induced by different drugs was examined by flow cytometry (FCM). Binding of HIF-1α to the MDR1 gene promoter was evaluated by Chromatin immunoprecipitation (ChIP). The relationship between HIF-1α/Pgp expression and sensitivity to chemotherapy was analyzed. Results: The sensitivity of LoVo MCS to all four chemotherapy drugs was decreased to varying degrees under hypoxic conditions. After silencing the HIF-1α gene, the sensitivities of LoVo MCS to all four chemotherapy drugs were restored. The apoptotic levels that all the drugs induced were all decreased to various extents in the hypoxic group. After silencing HIF-1α, the apoptosis level induced by all four chemotherapy drugs increased. The expression of HIF-1α and P-gp was significantly enhanced in LoVo MCS after treatment with hypoxia. Inhibiting HIF-1α significantly decreased the expression of MDR1/P-gp mRNA or protein in both the LoVo monolayers and LoVo MCS. The ChIP assay showed that HIF-1α was bound to the MDR1 gene promoter. Advanced colon carcinoma patients with expression of both HIF-1α and P-gp were more resistant to chemotherapy than that with non expression. Conclusions: HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-gp. The expression of HIF-1α and MDR1/P-gp can be used as a predictive marker for chemotherapy resistance in colon cancer. © 2014 Chen et al.

Figures

  • Figure 1. LoVo MCS show more resistance to chemotherapy than monolayers in hypoxia. (A) The morphology of LoVo monolayers. Scale bar = 50 mm. (B) The morphology of LoVo MCS. Scale bar = 50 mm. (C) Quantitative real-time PCR detected HIF-1a mRNA expression in LoVo MCS and in monolayers under normoxic and hypoxic conditions. The 18sRNA levels were used as internal control and the fold changes were calculated by the delta–delta Ct method. The experiments were performed in three biological replicates and PCR for each gene was done in duplicates (*p,0.05). (D) Drug sensitivities (mean 6 SD, n= 3) of LoVo MCS and LoVo monolayers to 5-FU in hypoxia or normoxia (*p,0.05, N: normoxia, H: hypoxia). doi:10.1371/journal.pone.0098882.g001
  • Figure 2. The sensitivity of different LoVo MCS groups to chemotherapy drugs was examined by MTT assay under normoxic and hypoxic conditions. (A) ADR. (B) VCR. (C) 5-FU. (D) CPT-11. (E) IC50 of different LoVo MCS groups to chemotherapeutics (mean 6 SD, n= 3, **p, 0.01, *p,0.05, N: normoxia, H: hypoxia). doi:10.1371/journal.pone.0098882.g002
  • Figure 3. The change in the proportion of annexin V-positive apoptotic cells under normoxic and hypoxic conditions after treatment with chemotherapy drugs. The proportion of annexin V-positive apoptotic cells(lower right quadrant) was evaluated by flow cytometry using annexin V allophycocyanin (APC) and propidium iodide (PI) staining of LoVo MCS after treatment with (B) ADR (25 mg/ml), (C) VCR (25 mg/ml), (D) 5-FU (200 mg/ml) and (E) CPT-11 (200 mg/ml). Untreated cells were used as negative control. Data of each statistical graph of Annexin V-APC/PI staining is expressed in % of cells in the lower right quadrant and represent the mean 6 SD of three independent experiments. The bar charts at the bottom of the flow cytometry scatterplots represent the % of cells undergoing apoptosis in the lower right quadrant (**p,0.01, N: normoxia, H: hypoxia). doi:10.1371/journal.pone.0098882.g003
  • Figure 4. Expression levels of HIF-1a and P-gp in LoVo MCS with LVV-HIF-1a (or MDR1) miR stably transduced. (A) Western blot analyses were performed. b-actin was used as internal control. (B) Relative protein levels (mean 6 SD, n = 3) of HIF-1a and P-gp were determined in each groups (**p,0.01, N: normoxia, H: hypoxia). doi:10.1371/journal.pone.0098882.g004
  • Figure 5. Expression levels of HIF-1a and MDR1/P-gp mRNA and protein in LoVo monolayers with LVV-HIF-1a miR stably transduced. (A, B) Quantitative real-time PCR detected HIF-1aor MDR1 mRNA expression in LoVo monolayers (hypoxia) with LVV-HIF-1a miR (A) or LVV-MDR1 miR (B) stably transduced. Non-treated monolayers in normoxia were used as normoxic control. The 18sRNA levels were used as internal control and the fold changes were calculated by the delta–delta Ct method. The experiments were performed in three biological replicates and PCR for each gene was done in duplicates (**p,0.01). (C–F) Western blot detected HIF-1a and P-gp protein expression in LoVo monolayers (hypoxia)with LVV-HIF-1amiR (C) or LVV-MDR1 miR (E) stably transduced. Non-treated monolayers in normoxia were used as normoxic control. (D, F) Comparison of expression levels (mean 6 SD, n= 3) of HIF-1a and MDR1 protein of each group (**p,0.01). doi:10.1371/journal.pone.0098882.g005
  • Figure 6. HIF-1a binds to the MDR1 and VEGF gene promoters in LoVo MCS. The binding of HIF-1a on the MDR1 and VEGF gene promoters was measured by ChIP. PCR analysis for the MDR1 gene promoter region was performed on immunoprecipitation samples (IP) with anti-HIF-1a antibody and with purified total input DNA from the LoVo MCS (normoxia and hypoxia). The VEGF gene promoter (an established HIF-1 target gene) was used as a positive control. Immunoprecipitation with non-specific IgG was performed as negative control. A sample representing linear amplification of the total input DNA was used as input control. doi:10.1371/journal.pone.0098882.g006
  • Table 1. The correlation between HIF-1a and P-gp expression and chemotherapy sensitivity.

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APA

Chen, J., Ding, Z., Peng, Y., Pan, F., Li, J., Zou, L., … Liang, H. (2014). HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-Glycoprotein. PLoS ONE, 9(6). https://doi.org/10.1371/journal.pone.0098882

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