Overcoming cisplatin resistance of ovarian cancer cells by targeting HIF-1-regulated cancer metabolism

Abstract

Cisplatin is currently one of the most effective chemotherapeutic drugs used for treating ovarian cancer; however, resistance to cisplatin is common. In this study, we explored an experimental strategy for overcoming cisplatin resistance of human ovarian cancer from the new perspective of cancer cell metabolism. By using two pairs of genetically matched cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines, we tested the hypothesis that downregulating hypoxia-inducible factor-1 (HIF-1), which regulates metabolic enzymes involved in glycolysis, is a promising strategy for overcoming cisplatin resistance of human ovarian cancer cells. We found that cisplatin downregulated the level of the regulatable α subunit of HIF-1, HIF-1α, in cisplatin-sensitive ovarian cancer cells through enhancing HIF-1α degradation but did not downregulate HIF-1α in their cisplatin-resistant counterparts. Overexpression of a degradation-resistant HIF-1α (HIF-1α ΔODD) reduced cisplatin-induced apoptosis in cisplatin-sensitive cells, whereas genetic knockdown of HIF-1α or pharmacological promotion of HIF-1α degradation enhanced response to cisplatin in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. We further demonstrated that knockdown of HIF-1α improved the response of cisplatin-resistant ovarian cancer cells to cisplatin by redirecting the aerobic glycolysis in the resistant cancer cells toward mitochondrial oxidative phosphorylation, leading to cell death through overproduction of reactive oxygen species. Our findings suggest that the HIF-1α-regulated cancer metabolism pathway could be a novel target for overcoming cisplatin resistance in ovarian cancer.

Keywords: Cancer metabolism; Cisplatin; HIF-1; Ovarian cancer; Resistance.

Figure 1. Cisplatin downregulates HIF-1α protein level and induces apoptosis in cisplatin-sensitive ovarian cancer cells but not in cisplatin-resistant ovarian cancer cells

(A) A2780, A2780/CP, PEO1, and PEO4 ovarian cancer cells were untreated or treated with the indicated concentrations of cisplatin for 72 h before being subjected to an MTT assay. The data shown are the OD values of treated cell groups at the end of treatment, expressed as a percentage of the OD value of the corresponding untreated cells. (B) A2780, A2780/CP, PEO1, and PEO4 ovarian cancer cells were treated with indicated concentrations of cisplatin for 24 h. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. (C) A2780, A2780/CP, PEO1, and PEO4 ovarian cancer cells were treated with 50 µM cisplatin for the indicated times. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. (D) A2780 and PEO1 ovarian cancer cells were treated with 50 µM cisplatin with or without 5 µM MG132 for the indicated times. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies.

Figure 2. Overexpression of a degradation-resistant HIF-1α reduces cisplatin-induced apoptosis in cisplatin-sensitive ovarian cancer cells

(A) and (B) A2780 and PEO1 ovarian cancer cells were transiently transfected with HIF-1α ΔODD plasmid or pcDNA3.1 control vector for 24 h. In (A), the cells were then either untreated or treated with 20 µM cisplatin for another 24 h. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. In (B), the cells were then treated with the indicated concentrations of cisplatin for 24 h and then subjected to an apoptosis ELISA. The bars indicating the standard deviations for apoptosis are not seen because they are smaller than the symbols on the lines, except for the 50 µM cisplatin dose in PEO1-pcDNA3.1 cells.

Figure 3. Genetic knockdown of HIF-1α expression restores sensitivity to cisplatin in cisplatin-resistant ovarian cancer cells

(A) and (B) A2780/CP and PEO4 ovarian cancer cells were transiently transfected with one of two different HIF-1α-specific siRNAs or control siRNA using Lipofectamine 2000 for 72 h. The cells were then either untreated or treated with 20 µM cisplatin for 24 h. In (A), cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. In (B), the cells were subjected to a live/dead cell viability assay and observed under a fluorescence microscope (scale bars, 200 µm). (C) A2780/CP and PEO4 cells were subjected to HIF-1α knockdown as described in (A). The cells were then either untreated or treated with 20 µM cisplatin for 72 h before being subjected to an MTT assay. The data shown are the OD values of treated cell groups at the end of treatment, expressed as a percentage of the OD value of the corresponding untreated cells.

Figure 4. Pharmacological promotion of HIF-1α degradation enhances sensitivity to cisplatin in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells

(A) The indicated ovarian cancer cells were treated with 20 µM cisplatin, 10 µM 1-methyl-1, 9 PA, or both for 24 h. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. (B) The indicated cells were treated as described in (A) for 36 h, and the cells were then harvested and subjected to flow cytometry analysis after staining with FITC-conjugated annexin V and propidium iodide. (C) The indicated cells were treated as described in (A) for 24 h and then subjected to a live/dead cell viability assay and observed under a fluorescence microscope (scale bars, 200 µm). (D) The indicated cells were treated as described in (A) for 72 h before being subjected to an MTT assay. The data shown are the OD values of treated cell groups at the end of treatment, expressed as a percentage of the OD value of the corresponding untreated cells. (E) A2780/CP and PEO4 ovarian cancer cells were transiently transfected with HIF-1α ΔODD plasmid or pcDNA3.1 control vector for 24 h. After the transfection, the cells were treated with 20 µM cisplatin, 10 µM 1-methyl-1, 9 PA, or both for 24 h as described in (A). Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies.

Figure 5. Cisplatin plus HIF-1α downregulation induces apoptosis of cisplatin-resistant ovarian cancer cells through inducing ROS overproduction

(A) The indicated ovarian cancer cells were treated with 20 µM cisplatin, 10 µM 1-methyl-1, 9 PA, or both for 24 h. The cells were then stained with Enzo’s ROS detection kit and observed under a fluorescence microscope (scale bars, 100 µm). (B) A2780/CP and PEO4 ovarian cancer cells were transiently transfected with one of two different HIF-1α-specific siRNAs or control siRNA using Lipofectamine 2000 for 72 h. The cells were then either untreated or treated with 20 µM cisplatin for 24 h. The cells were then stained with Enzo’s ROS detection kit and observed under a fluorescence microscope (scale bars, 100 µm). (C) Top, A2780 and PEO1 ovarian cancer cells were treated with 20 µM cisplatin with or without 5 mM NAC for 24 h. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. Bottom, the cells were treated with 20 µM cisplatin with or without 5 mM NAC for 72 h before being subjected to MTT assay. The data shown are the OD values of treated cell groups at the end of treatment, expressed as a percentage of the OD value of the corresponding untreated cells. (D) Top, A2780/CP and PEO4 cells were treated with 20 µM cisplatin, 10 µM 1-methyl-1, 9 PA, or both, in the presence or absence of 5 mM NAC for 24 h. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. Bottom, the cells were treated as described above for 72 h before being subjected to MTT assay as in (C).

Figure 6. Apoptosis induced by cisplatin plus HIF-1α downregulation can be partially reduced by overexpression of LDH-A

(A) A2780/CP and PEO4 ovarian cancer cells were transiently transfected with one of two different HIF-1α-specific siRNAs or control siRNA using Lipofectamine 2000 for 72 h. The cells were untreated or treated with 20 µM cisplatin for 24 h. Cell lysates were then prepared and subjected to Western blotting analysis using the indicated antibodies. (B) and (C) A2780 and PEO1 ovarian cancer cells were infected with a pLEX-based recombinant lentivirus containing human LDH-A cDNA or not for 24 h. The cells were then transiently transfected with each of two different HIF-1α-specific siRNAs or control siRNA using Lipofectamine 2000 for 72 h. In the last 24 h of siRNA treatment, the cells were exposed to 20 µM cisplatin or not. After the treatment, cell lysates were prepared and subjected to Western blotting analysis using the indicated antibodies (B) or subjected to apoptosis ELISA (C).