It is well known that hypoxia prospects to numerous effects in invasive and metastatic cancers including a shift toward anaerobic glycolysis, away from the oxidative mitochondrial phosphorylation. unique metabolites representing numerous metabolite classes including carbohydrates, amino acids, carboxylic acids and nucleotides. BPTES induced metabolism changes in the malignancy cell lines were especially pronounced under hypoxic conditions with up to 1/3 of the metabolites altered significantly (< 0.05) relative to untreated cells. The BPTES induced changes were more pronounced for MCF7 cells, with 14 metabolites altered significantly (< 0.05) compared to seven for MDA-MB231. Analyses of the results show that BPTES affected numerous metabolic pathways including glycolysis, TCA cycle, nucleotide and amino acid metabolism in cancer. The distinct metabolic responses to BPTES treatment determined in the two breast cancer cell lines offer valuable metabolic information for the exploration of the therapeutic responses to breast cancer. = 3) and hypoxic (= 3) conditions were treated with 20 M BPTES inhibitor (Sigma-Aldrich St. Louis, MO) before incubation. Metabolite extraction After 24 h incubation, the cell media were removed and the cells washed with 30 mL cold water; a mixture of methanol/chloroform (9.5 mL; 9:1 v/v) was then immediately added to the plates to quench the cells and extract metabolites. Cell lysates were obtained by keeping the plates at ?75C for 5 min and thawing them at room temperature. Cell remnants were scraped from the culture dishes and collected in fresh tubes along with the cell lysates. Resulting mixtures were centrifuged at 13,000 rpm for 5 min and supernatant solutions that contained cell metabolites were transferred to fresh tubes and dried overnight using a Speedvac at 30C. The dried residues were dissolved in 600 L 0.1 M phosphate buffer (pH 7.4) in D2O solvent containing 50 M TSP and the solutions transferred to 5 mm NMR tubes for metabolite analysis using 1H 1D NMR spectroscopy. Metabolite labeling with a 15N- isotope tag After acquiring 1H 1D NMR spectra as described below for cell extracts, the solutions were dried and reconstituted in 550 L water. Carboxyl group containing metabolites were then labeled with 15N-cholamine (Figure ?(Figure1),1), which was synthesized using a two-step reaction following the protocol described previously by our laboratory (Tayyari et al., 2013). Briefly, 15N-cholamine (5 mg, 50 mol) was added to solutions of cell extracts in Eppendorf tubes and pH adjusted to 7.0 CCG 50014 with 1 M hydrochloric acid (HCl) or sodium hydroxide (NaOH). DMTMM (15 mg) was then added as a catalyst to help initiate the reaction, and the mixtures were then stirred at room temperature for 4 h to complete the reaction. The resulting solutions were mixed with a small volume (25 L) of D2O for NMR field-frequency locking. To maintain amide protonation the pH was adjusted to 5.0 by adding 1 N HCl or 1 N NaOH. The solutions were then transferred to 5 mm NMR tubes for detection of the isotope tagged metabolites using two-dimensional NMR spectroscopy. Open in a separate window Figure 1 (A) General reaction for tagging of carboxylic group containing metabolites with 15N-cholamine tag; (B) schematic 3D view of a typical 2D 1H-15N HSQC NMR spectrum of a sample with 15N-cholamine tagging of carboxylic acid containing metabolites. DMTMM:4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium chloride. NMR spectroscopy All NMR experiments were performed at 298 K on a Bruker Avance III 800 MHz spectrometer equipped with a cryoprobe and Z-gradients. Before labeling with the cholamine tag, 1H 1D NMR experiments were performed on the cell extracts using the CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence with residual water signal suppression using presaturation. A spectral width of 9,615 Hz, time domain points of 32 K, a recycle delay of 6 s, 16 dummy scans and 64 scans were used. The raw data were then Fourier transformed after multiplying with an exponential window function using a line broadening (LB) of 0.5 Hz and spectrum size of 32 K points. Resulting 1D spectra were phase and baseline corrected. To detect the carboxyl group containing metabolites after isotope tagging, sensitivity-enhanced 1H-15N 2D HSQC experiments were performed with an INEPT transfer delay of 6 ms corresponding to the 1JNH coupling of 90 Hz. Spectral widths for the 1H and 15N dimensions were approximately 8 and 3 kHz, respectively. One hundred and twenty-eight free induction decays of 1 1,024 data points each were collected in the indirect dimension (t1) with 16 transients per increment. 15N decoupling during the direct acquisition dimension (t2) Slc4a1 was achieved with the GARP (Globally Optimized Alternating-Phase Rectangular Pulses) sequence. The resulting 2D data were zero-filled to 2,048 points in the t2 and 1,024 in the t1 dimension after forward linear prediction to 256 points. A 90 shifted squared sine-bell window function was CCG 50014 applied to both dimensions before Fourier transformation. Chemical shifts were referenced to the TSP signal for 1H 1D NMR or the CCG 50014 derivatized formic acid signal (1H: 8.05 ppm; 15N: 123.93 ppm) in.