Adrenomedullin induces cisplatin chemoresistance in ovarian cancer through reprogramming of glucose metabolism

Materials

Cisplatin-sensitive A2780 EOC cells and cisplatin-resistant A2780cis cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC; Porton Down, Salisbury, UK). Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Sigma Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Recombinant human ADM (full length 1–53 amino acids) and its receptor antagonist ADM22-52 were purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). Mouse-anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). IRDye-conjugated affinity-purified anti-rabbit and anti-mouse IgGs were purchased from Rockland (Gilbertsville, PA, USA). Trizol reagent and the reverse transcription (RT) system were purchased from Invitrogen Inc. (Grand Island, NY, USA). All other chemicals and drugs were purchased from Sigma Chemical (St. Louis, MO, USA).

Cell culture

Cisplatin-sensitive A2780 cells and cisplatin-resistant A2780cis cells were cultured in RPMI 1640 containing 10% FBS and penicillin/ streptomycin (100 U/mL) in a humidified 37℃ incubator. When confluent, cells were treated with ADM. For the inhibition experiments, cells were pretreated with ADM22-52 for 1 h before stimulation with ADM.

Construction of expression vector and cell transfection

The short hairpin (sh)RNA sequences targeting ADM or scramble sequences were designed as previously reported.^[22] The shRNA sequences were submitted to BLAST (http://www.ncbi.nlm.nih.gov/blast/) to ensure their specificity of targeting. The pRNA-U6.1/Neo was selected as the RNAi expression vector. The double-stranded shDNA was annealed, ligated into the restriction sites between BamHI and XhoI in pRNA-U6.1/Neo with T4 DNA ligase, and then transfected into Escherichia coli DH5α. Resistance screening, agarose gel electrophoresis, and DNA sequencing were used to check the recombinant plasmid, named pRNA-shADM.

For transfection, cells (5 × 104) were plated in six-well plates and allowed to adhere for 24 h. Plasmid DNA was transfected into cells using Lipofectamine^™ 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cells were transfected using 2 μg DNA mixed with 5 μL Lipofectamine in 1 mL medium without serum or antibiotics. The cells were incubated for 24 h, and 1 mL medium containing 20% serum was added to each well. The cells were cultured and screened in medium containing 10% serum and 400 μg/mL G418 for at least 3 weeks. Stable transfectants were formed.

Cell proliferation detected with Cell Counting Kit-8

The Cell Counting Kit‑8 (CCK‑8) assays were used to determine the effect of ADM on SKOV3 cell viability. Cells (103/well) were incubated in 96‑well plates overnight, starved in serum‑free medium for 24 hm and treated with indicated reagents. Cells were incubated with 10 μL CCK-8 solution (Dojindo Molecular Technologies, Kumamoto, Japan) for 1 h and the absorbance was measured at 450 nm.

RNA extraction and reverse transcription-polymerase chain reaction analysis

Total RNA was isolated using TRIzol reagent. Total RNA (2 μg) was reverse transcribed using an RT system. One microliter of the reaction mixture was subjected to reverse transcription-polymerase chain reaction (RT-PCR). The polymerase chain reaction (PCR) primers are listed in Table 1. All amplification reactions were performed under the following conditions: 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 60 s. For the quantitative real-time RT-PCR analysis, the amount of PCR products formed in each cycle was evaluated on the basis of SYBR Green I (Solarbio, Beijing, China) fluorescence. Results were analyzed with Stratagene Mx3000 software, and mRNA levels were normalized with respect to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each sample. The primers used in this study were as follows: human ADM, 5′-TCCCCCTATTTTAAGACGTGAATG-3′ and 5′-CATGCACACAAACACACTCACAT-3′and human GAPDH, 5′- GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA-3′.

Preparation of cytosolic proteins and western blotting

Following treatment, the cells were packed by centrifugation at 200 ×g for 3 min and homogenized in ice-cold fractionation buffer. The cell lysate was incubated on ice for 15 min and then centrifuged at 20,000 ×g for 30 min at 4°C. The cytosolic fraction was collected and subjected to 10% SDS-PAGE. Protein concentrations were determined by BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The proteins were transferred to a polyvinylidene fluoride membrane. The membrane was incubated successively with 5% bovine serum albumin in Tris Tween buffered saline at room temperature for 1 h, followed by incubation overnight at 4°C with the individual primary antibody. Specific reaction was detected using IRDye-conjugated second antibody and visualized using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Quantification of image density in pixels was performed using the Odyssey infrared imaging system (LI-COR Biosciences).

Apoptosis analysis

Cells were cultured in 96-well plates with or without ghrelin (10^-9 mol/L) stimulation. Apoptosis was assessed by measuring cysteine aspartic acid-specific protease (caspase) 3/7 activity with the use of the Caspase-Glo^® 3/7 Assay Kit (Promega, Madison, WI, USA).

Extracellular flux analysis

Oxygen consumption rate (OCR) and extracellular acidification rates (ECARs) were measured on a Seahorse XF24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA). Cells were seeded into a Seahorse 24-well plate coated with poly-l-lysine hydrobromide. Before assay, the cells were incubated with Seahorse XF base medium supplemented with 10 mmol/L glucose, 1 mmol/L pyruvate, and 2 mmol/L glutamine in a CO²-free incubator equilibrated for 1 h. During assay, 1 mmol/L oligomycin, 1 mmol/ L carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 1 mmol/L rotenone, and 1 mmol/L antimycin A were added sequentially. The data were analyzed with Wave software and the XF Mito/Glycolysis Stress Test Report Generator (Seahorse Bioscience).

Detection of lactate in cell culture medium

The cells were inoculated into the 24-well plates at 1 × 105/ well. Each group had five duplicated wells. The cell culture medium was collected, and then, the level of lactate was measured according to the operation instructions of the enzyme-linked immunosorbent assay (ELISA) kit (Keshun Bio, China).

Statistical analysis

Quantitative data are presented as the means ± standard error of mean (SEM) determined from the indicated number of experiments. Statistical analysis was based on Student’s t-test for comparison of two groups or one-way analysis of variance for multiple comparisons. P < 0.05 was used to determine statistical significance.

ADM expression is upregulated in cisplatin-resistant EOC cells

We examined the expression of ADM in epithelial ovarian carcinoma cells. Compared to cisplatin-sensitive control A2780 cells, the levels of ADM mRNA in cisplatin-resistant A2780cis cells increased significantly (Figure 1A). A similar increment was observed for ADM at the protein level (Figure 1B). These results confirmed that ADM expression was upregulated in cisplatin-resistant cells.

ADM inhibited cisplatin chemosensitivity of EOC cells

We confirmed the cisplatin chemosensitivity of EOC cells.

Figure 1

Expression of ADM in ovarian epithelial carcinoma cells. (A) ADM mRNA expression and (B) ADM protein levels in cisplatin-sensitive control A2780 cells and cisplatin-resistant A2780cis cells. GAPDH was used as an internal control. Data are means ± SEM. ADM: adrenomedullin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SEM: standard error of the mean.

The survival rate of A2780 cells significantly decreased under cisplatin treatment at 5–40 μmol/L, whereas the cell survival rate of A2780cis cells significantly decreased under cisplatin treatment at 20–40 μmol/L (Figure 2A). We chose 5 and 20 μmol/L cisplatin for treatment of A2780 and A2780cis cells. Inhibition of cell survival by cisplatin treatment was significantly attenuated by ADM (Figure 2B) in cisplatin-sensitive A2780 cells, as well as apoptosis (Figure 2C), which was attenuated by ADM22-52, the receptor competitor of ADM, indicating inhibition of cisplatin chemosensitivity. In cisplatin-resistant A2780cis cells, knockdown of ADM significantly abolished the resistance to cisplatin (Figure 2D), as well as increasing apoptosis (Figure 2E and F). These results indicated that ADM inhibited cisplatin chemosensitivity of EOC cells through its receptor.

ADM induced reprogramming of glucose metabolism in EOC cells

Emerging evidence has found that tumor cells can rapidly switch glucose metabolism to aerobic glycolysis, a phenomenon known as the Warburg effect, to rapidly provide ATP and metabolic intermediates for supporting proliferation. Lactate secretion is one part of the classical glycolytic index. We found significantly higher level of lactate in the medium of A2780cis cells (Figure 3A), indicating increased glycolysis in cisplatin-resistant cells. ADM induced secretion of lactate in EOC medium (Figure 3B). Knockdown of ADM significantly abolished glycolysis in A2780cis cells (Figure 3C). Similar results were found for change of cellular ECAR (indicative of glycolysis), but not OCR (indicative of oxidative phosphorylation) (Figure 3D and E). These results indicated that ADM induced reprogramming of glucose metabolism in EOC cells.

Figure 2

Effect of ADM on cisplatin chemosensitivity of EOC cells. (A) Cisplatin-sensitive control A2780 cells and cisplatin-resistant A2780cis cells were treated with cisplatin (DDP) supplied for 72 h, and viability was detected using CCK‑8 assays. (B) Sensitive A2780 cells were treated with cisplatin (5 μmol/L) and ADM for 72 h, and viability was detected using CCK‑8 assays. (C) Caspase-3/7 activity detected in A2780 cells treated with ADM. Cells were pretreated with ADM22-52 for 1 h and then with ADM (10^-8 mol/L) for 72 h. Data are means ± SEM from four separate experiments. ^*P < 0.05 versus control; ^#P < 0.01 versus cisplatin treatment alone. (D–F) ADM was knocked down in A2780cis cells through transfection with ADM-targeted recombinant plasmid (pRNA-shADM) or control (pRNA-U6.1/Neo), and viability (D), caspase-3/7 activity (E), cleaved caspase-3 and Bcl-2 protein levels (F) were detected. Data are means ± SEM from four separate experiments. ^*P < 0.05 versus PBS treatment; ^#P < 0.01 versus pRNA-U6.1/Neo transfection. ADM: adrenomedullin; EOC: epithelial ovarian cancer; PBS: phosphate buffer solution; CCK-8: Cell Counting Kit‑8; SEM: standard error of the mean.

Figure 3

Role of ADM in aerobic glycolysis in EOC cells. (A) Increased lactate levels (indicators of glycolysis) in cisplatin-resistant A2780cis cells. (B) Effects of ADM on lactate levels in A2780 cells. (C) Knockdown of ADM significantly abolished glycolysis in A2780cis cells. (D) ADM increased ECAR levels (indicator of glycolysis), but not OCR levels (indicator of oxidative phosphorylation) in A2780 cells. (E) Knockdown of ADM significantly decreased ECAR levels in A2780cis cells. All data are shown as mean ± SEM (n = 4). ^*P < 0.05 versus control; ^#P < 0.01 versus ADM treatment. ADM: adrenomedullin; EOC: epithelial ovarian cancer; PBS: phosphate buffer solution; OCR: oxygen consumption rate; ECAR: extracellular acidification rate; SEM: standard error of the mean.

Reprogramming of glucose metabolism mediated ADM-induced cisplatin chemoresistance in EOC cells

Previous studies have found that the activation of pyruvate kinase isoenzyme M2 (PKM2) is involved in the aerobic glycolysis of tumor cells. The protein level of PKM2 is significantly higher in cisplatin-resistant A2780cis cells, indicating the key role of glucose metabolism reprogramming in EOC chemosensitivity (Figure 4A). Exogenous administration of ADM significantly increased PKM2 protein level (Figure 4B), which was abolished by ADM22-52; knockdown of endogenous ADM significantly reduced PKM2 protein level (Figure 4C), indicating that PKM2 is the downstream molecule of ADM signaling. In cisplatin-sensitive A2780 cells, compound 3k, a PKM2 inhibitor, significantly abolished the ADM-improved cell survival (Figure 4D) and ADM-inhibited apoptosis (Figure 4E and F), whereas DASA, the PKM2 activator, significantly restored cell survival (Figure 4G) and inhibited apoptosis (Figure 4H and I) induced by lack of endogenous ADM. These results showed that reprogramming of glucose metabolism mediated ADM-induced cisplatin chemoresistance in EOC cells.