Gastric cancer depends on aldehyde dehydrogenase 3A1 for fatty acid oxidation

The major source of ATP in cancer cells remains unclear. Here, we examined energy metabolism in gastric cancer cells and found increased fatty acid oxidation and increased expression of ALDH3A1. Metabolic analysis showed that lipid peroxidation by reactive oxygen species led to spontaneous production of 4-hydroxynonenal, which was converted to fatty acids with NADH production by ALDH3A1, resulting in further fatty acid oxidation. Inhibition of ALDH3A1 by knock down using siRNA of ALDH3A1 resulted in significantly reduced ATP production by cancer cells, leading to apoptosis. Oxidative phosphorylation by mitochondria in gastric cancer cells was driven by NADH supplied via fatty acid oxidation. Therefore, blockade of ALDH3A1 together with mitochondrial complex I using gossypol and phenformin led to significant therapeutic effects in a preclinical gastric cancer model.

After 24 h, transfected cells were seeded in XF cell culture microplates at a density of 30,000/well and incubated for 48 h at 37°C. To measure the oxygen consumption rate (OCR), cells were incubated in XF base medium supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, and then equilibrated in a non-CO2 incubator for 1 h before starting the assay. Samples were mixed (3 min) and measured (3 min) in an XFe96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, Billerica, MA, U.S.A.). Oligomycin (1 µM), carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP, 1 µM), and rotenone/antimycin A (0.5 µM) were injected at the indicated times. Finally, the OCR was normalized in a SRB assay.

Sample preparation
One million cells were harvested in 1 mL cold methanol after sequential washing with PBS.
Next, cells were lysed by vigorous vortexing, acidified with HCl (final concentration, 25 mM), and then mixed with 50 μL internal standard (myristic acid-d27; 0.1 mg/mL). The sample was centrifuged at 13000 rpm for 10 min and the supernatant was collected in a fresh tube. Next, 3 mL iso-octane was added and the tube was centrifuged at 4000 rpm for 20 min. Finally, the upper layer was collected and dried under vacuum.

Fatty acid methyl ester (FAME) derivatization
The dried sample was reacted with 200 μL BCl3-MeOH (12% w/w; Sigma-Aldrich) at 60°C for 30 min. Next, 100 μL H2O and 100 μL hexane were added sequentially and mixed vigorously. The upper phase was collected after resting for 5 min. Then, 20-30 mg anhydrous sodium sulfate was added prior to GC/MS analysis. FAMEs (Sigma-Aldrich) were used to generate calibration curves without derivatization.

GC-MS
FAMEs were analyzed in a GC-MS system (Agilent7890A/5975C) fitted with a capillary column (HP-5MS; 30 m × 0.25 mm × 0.2 µm). Electron impact ionization was used in positive ion mode, with an injection volume of 1 μL and a split mode ratio of 10:1. Total analysis time was 73.7 min and the temperature gradient was as follows: hold at 50°C for 2 min; 50°C to 120°C at 10°C /min; 120°C to 250°C at 3°C /min; hold at 250°C for 15 min; 250°C to 300°C at 35°C /min; hold at 300 for 5 min. The calibration range was 0.001-10 mg/mL (r2≥0.99). Data analysis was performed using MSD Chemstation software (Agilent E02.02.1431).

Sample preparation
One million cells were harvested using 1.4 mL of cold methanol/H2O (80/20, v/v) after sequential washing with PBS and H2O. Cells were lysed by vigorous vortexing prior to addition of 100 μL internal standard (Malonyl-13C3 CoA; 5 μM). Chloroform was added and metabolites were extracted from the aqueous phase by liquid-liquid extraction. The aqueous phase was dried in a vacuum centrifuge and the sample was reconstituted with 50 μL of H2O/MeOH (50/50 v/v) prior to LC-MS/MS analysis.
Next, 3 μL sample was injected into the LC-MS/MS system and ionized by a turbo spray ionization source. Acetonitrile/H2O (10/90) with 15 mM ammonium hydroxide and acetonitrile with 15 mM ammonium hydroxide were used as mobile phase A and B, respectively. The separation gradient was as follows: hold at 0% B for 3 min; 0% to 50% B for 2 min; 50% to 70% B for 5 min; 70% to 0% B for 0.1 min; hold at 0% B for 4.9 min. LC flow was 200 μL/min and the column temperature was kept at 25°C. Multiple reaction monitoring was used in positive ion mode and the extracted ion chromatogram corresponding to the specific transition for each fatty acyl CoA was used for quantitation.
The calibration range for fatty acyl CoAs was 0.1-10000 nM (r2≥0.99). Data analysis was performed using Analyst 1.5.2 software.

Cell cycle analysis
Cells were incubated with or without ALDH3A1 siRNA for 48 h, or with gossypol (5 μM) and/or phenformin (100 μM) for 24 h. Cells were then collected, washed twice with PBS, centrifuged at 1500 rpm for 5 min, and fixed overnight at 4°C with 70% ethanol. They were then centrifuged at 2000 rpm for 10 min, stained with PI + RNase solution for 30 min in the dark, washed with cold PBS, and then analyzed in a FACSCalibur flow cytometer (BD Falcon, Bedford, MA, USA).